NISHIYAMA Masayoshi

Researcher Information

Degree

• Doctor(Science)（Osaka University）

Research funding number

• 10346075

J-Global ID

• 200901058966412166

Research Interests

• 生命動態物理学   Mechanobiology   Molecular Machinery   Cell Motility   High-pressure microscope   Dynamics of Biomolecules   Single Molecule Detection   

Research Areas

• Life sciences / Biophysics
• Nanotechnology/Materials / Nanobioscience
• Nanotechnology/Materials / Nanomaterials

Education

• 1996/04 - 1998/03  Osaka University  基礎工学研究科 物理系専攻 生物工学分野

Association Memberships

• The Japanese Society of Mechanobiology   THE JAPAN SOCIETY OF APPLIED PHYSICS   The Biophysical Society of Japan   Biophysical Society   THE JAPAN SOCIETY OF HIGH PRESSURE SCIENCE AND TECHNOLOGY   

Published Papers

• Compounding deep sea physical impacts on marine microbial motility

  Kelli K. Mullane; Masayoshi Nishiyama; Tatsuo Kurihara; Douglas H. Bartlett

  Frontiers in Marine Science Frontiers Media SA 10 1181062  2023/05 [Refereed]
   

  Introduction Approximately three-fourths of all pelagic marine prokaryotes live in the deep sea, an environment characterized by high hydrostatic pressure and, in most cases, low temperature. Labile organic matter is often scarce within these settings, providing a competitive advantage to motile cells that can access the nutrients within a greater seawater volume. Because many cells present at depth are shallow water-adapted microbes descending from more productive surface waters, deep-sea conditions could significantly reduce their motility and, consequently, their biogeochemical activities. Methods In this study, we address this possibility by examining the impact of deep-sea physical conditions on the motility of three representative marine microbes belonging to the cosmopolitan genera Halomonas, Alcanivorax, and Shewanella. Growth-dependent motility agar assays and growth-independent microscopy assays were employed at four pressures and two temperatures. Results At pressures equivalent to bathyal and abyssal depths (10 – 50 Megapascals), decreases in temperature (30°C – 4°C or 23°C – 7°C depending on the assay) had a greater negative impact on motility than pressure. In addition, the high-pressure and low-temperature impacts were additive. Exposure to high pressure and/or low temperature had varying degrees of effect on flagellar function, depending on the strain and the magnitude of the applied stress. These ranged from short-term impacts that were quickly reversible to long-term impacts that were detrimental to the function of the flagellum, leading to complete loss of motility. Discussion These findings highlight the sensitivity of motility systems of piezosensitive mesophilic marine bacteria to the combined pressure/temperature conditions present in the deep sea, phenotypes that in situ are likely to manifest themselves in the modulation of diverse microbial activities.
• High hydrostatic pressure induces slow contraction in mouse cardiomyocytes.

  Yohei Yamaguchi; Masayoshi Nishiyama; Hiroaki Kai; Toshiyuki Kaneko; Keiko Kaihara; Gentaro Iribe; Akira Takai; Keiji Naruse; Masatoshi Morimatsu

  Biophysical journal 2022/07 [Refereed]
   

  Cardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.
• Activation of Eukaryotic Flagellar Beating at High-Pressure

  Toshiki YAGI; Masayoshi NISHIYAMA

  The Review of High Pressure Science and Technology The Japan Society of High Pressure Science and Technology 31 (2) 66 - 73 0917-639X 2021/10 [Refereed][Invited]
  
• Pressure-induced changes on the morphology and gene expression in mammalian cells

  Kazuko Okamoto; Tomonobu M; Watanabe; Masanobu Horie; Masayoshi Nishiyama; Yoshie Harada; Hideaki Fujita

  Biology Open 10 (7) 1 - 7 2021/07 [Refereed]
  
• Glycine insertion modulates the fluorescence properties of Aequorea victoria green fluorescent protein and its variants in their ambient environment

  Takamitsu J Morikawa; Masayoshi Nishiyama; Keiko Yoshizawa; Hideaki Fujita; Tomonobu M Watanabe

  Biophysics and Physicobiology 18 145 - 158 2021/03 [Refereed]
  
• A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation.

  Yosuke Toyotake; Masayoshi Nishiyama; Fumiaki Yokoyama; Takuya Ogawa; Jun Kawamoto; Tatsuo Kurihara

  Biomolecules MDPI AG 10 (5) 745 - 745 2020/05 [Refereed]
   

  Lysophosphatidic acid acyltransferase (LPAAT) introduces fatty acyl groups into the sn-2 position of membrane phospholipids (PLs). Various bacteria produce multiple LPAATs, whereas it is believed that Escherichia coli produces only one essential LPAAT homolog, PlsC-the deletion of which is lethal. However, we found that E. coli possesses another LPAAT homolog named YihG. Here, we show that overexpression of YihG in E. coli carrying a temperature-sensitive mutation in plsC allowed its growth at non-permissive temperatures. Analysis of the fatty acyl composition of PLs from the yihG-deletion mutant (∆yihG) revealed that endogenous YihG introduces the cis-vaccenoyl group into the sn-2 position of PLs. Loss of YihG did not affect cell growth or morphology, but ∆yihG cells swam well in liquid medium in contrast to wild-type cells. Immunoblot analysis showed that FliC was highly expressed in ∆yihG cells, and this phenotype was suppressed by expression of recombinant YihG in ∆yihG cells. Transmission electron microscopy confirmed that the flagellar structure was observed only in ∆yihG cells. These results suggest that YihG has specific functions related to flagellar formation through modulation of the fatty acyl composition of membrane PLs.
• Session 1SHA-control of biological functions with hydrostatic pressure stimulation.

  Hiroaki Hata; Masayoshi Nishiyama

  Biophysical reviews 12 (2) 269 - 270 2020/04 [Refereed]
  
• Increased hydrostatic pressure induces nuclear translocation of DAF-16/FOXO in C. elegans.

  Naoshi Watanabe; Masatoshi Morimatsu; Ayano Fujita; Mika Teranishi; Surabhi Sudevan; Masaru Watanabe; Hiroaki Iwasa; Yutaka Hata; Hiroyuki Kagi; Masayoshi Nishiyama; Keiji Naruse; Atsushi Higashitani

  Biochemical and biophysical research communications 523 (4) 853 - 858 2020/03 [Refereed]
   

  Mechanical stimulation is well known to be important for maintaining tissue and organ homeostasis. Here, we found that hydrostatic pressure induced nuclear translocation of a forkhead box O (FOXO) transcription factor DAF-16, in C. elegans within minutes, whereas the removal of this pressure resulted in immediate export of DAF-16 to the cytoplasm. We also monitored DAF-16-dependent transcriptional changes by exposure to 1 MPa pressure for 5 min, and found significant changes in collagen and other genes in a DAF-16 dependent manner. Lifespan was markedly prolonged with exposure to cyclic pressure treatment (1 MPa once a day for 5 min from L1 larvae until death). Furthermore, age-dependent decline in locomotor activity was suppressed by the treatment. In contrast, the nuclear translocation of the yes-associated protein YAP-1 was not induced under the same pressure conditions. Thus, moderate hydrostatic pressure improves ageing progression through activation of DAF-16/FOXO in C. elegans.
• High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration.

  Hiroaki Hata; Yasutaka Nishihara; Masayoshi Nishiyama; Yoshiyuki Sowa; Ikuro Kawagishi; Akio Kitao

  Scientific reports 10 (1) 2351 - 2351 2020/02 [Refereed]
   

  High pressure below 100 MPa interferes inter-molecular interactions without causing pressure denaturation of proteins. In Escherichia coli, the binding of the chemotaxis signaling protein CheY to the flagellar motor protein FliM induces reversal of the motor rotation. Using molecular dynamics (MD) simulations and parallel cascade selection MD (PaCS-MD), we show that high pressure increases the water density in the first hydration shell of CheY and considerably induces water penetration into the CheY-FliM interface. PaCS-MD enabled us to observe pressure-induced dissociation of the CheY-FliM complex at atomic resolution. Pressure dependence of binding free energy indicates that the increase of pressure from 0.1 to 100 MPa significantly weakens the binding. Using high-pressure microscopy, we observed that high hydrostatic pressure fixes the motor rotation to the counter-clockwise direction. In conclusion, the application of pressure enhances hydration of the proteins and weakens the binding of CheY to FliM, preventing reversal of the flagellar motor.
• High hydrostatic pressure induces vigorous flagellar beating in Chlamydomonas non-motile mutants lacking the central apparatus.

  Toshiki Yagi; Masayoshi Nishiyama

  Scientific reports 10 (1) 2072 - 2072 2020/02 [Refereed]
   

  The beating of eukaryotic flagella (also called cilia) depends on the sliding movements between microtubules powered by dynein. In cilia/flagella of most organisms, microtubule sliding is regulated by the internal structure of cilia comprising the central pair of microtubules (CP) and radial spokes (RS). Chlamydomonas paralyzed-flagella (pf) mutants lacking CP or RS are non-motile under physiological conditions. Here, we show that high hydrostatic pressure induces vigorous flagellar beating in pf mutants. The beating pattern at 40 MPa was similar to that of wild type at atmospheric pressure. In addition, at 80 MPa, flagella underwent an asymmetric-to-symmetric waveform conversion, similar to the one triggered by an increase in intra-flagella Ca2+ concentration during cell's response to strong light. Thus, our study establishes that neither beating nor waveform conversion of cilia/flagella requires the presence of CP/RS in the axoneme.
• Molecular dynamics simulation of proteins under high pressure: Structure, function and thermodynamics.

  Hiroaki Hata; Masayoshi Nishiyama; Akio Kitao

  Biochimica et biophysica acta. General subjects 1864 (2) 129395 - 129395 0304-4165 2020/02 [Refereed]
   

  BACKGROUND: Molecular dynamics (MD) simulation is well-recognized as a powerful tool to investigate protein structure, function, and thermodynamics. MD simulation is also used to investigate high pressure effects on proteins. For conducting better MD simulation under high pressure, the main issues to be addressed are: (i) protein force fields and water models were originally developed to reproduce experimental properties obtained at ambient pressure; and (ii) the timescale to observe the pressure effect is often much longer than that of conventional MD simulations. SCOPE OF REVIEW: First, we describe recent developments in MD simulation methodologies for studying the high-pressure structure and dynamics of protein molecules. These developments include force fields for proteins and water molecules, and enhanced simulation techniques. Then, we summarize recent studies of MD simulations of proteins in water under high pressure. MAJOR CONCLUSIONS: Recent MD simulations of proteins in solution under pressure have reproduced various phenomena identified by experiments using high pressure, such as hydration, water penetration, conformational change, helix stabilization, and molecular stiffening. GENERAL SIGNIFICANCE: MD simulations demonstrate differences in the properties of proteins and water molecules between ambient and high-pressure conditions. Comparing the results obtained by MD calculations with those obtained experimentally could reveal the mechanism by which biological molecular machines work well in collaboration with water molecules.
• Tree of motility - A proposed history of motility systems in the tree of life.

  Makoto Miyata; Robert C Robinson; Taro Q P 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 : devoted to molecular & cellular mechanisms 25 (1) 6 - 21 2020/01 [Refereed]
   

  Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.
• Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas.

  Sotaro Fujii; Misa Masanari-Fujii; Shinya Kobayashi; Chiaki Kato; Masayoshi Nishiyama; Yoshie Harada; Satoshi Wakai; Yoshihiro Sambongi

  Bioscience, biotechnology, and biochemistry 1 - 8 0916-8451 2018/03 [Refereed]
   

  Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1-150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.
• 細菌の祖先がもつ運動マシナリーを現代に蘇らせる (特集 運動マシナリーの多様性から見えるもの(前編))

  西山 雅祥; 金井 保; 竹川 宜宏

  生物工学会誌 日本生物工学会 96 (4) 191 - 194 0919-3758 2018
• Morphological Control of Microtubule-Encapsulating Giant Vesicles by Changing Hydrostatic Pressure.

  Kingo Takiguchi; Masahito Hayashi; Yuki Kazayama; Taro Toyota; Yoshie Harada; Masayoshi Nishiyama

  Biological & pharmaceutical bulletin 41 (3) 288 - 293 0918-6158 2018 [Refereed]
   

  For the development of artificial cell-like machinery, liposomes encapsulating cytoskeletons have drawn much recent attention. However, there has been no report showing isothermally reversible morphological changes of liposomes containing cytoskeletons. We succeeded in reversibly changing the shape of cell-sized giant vesicles by controlling the polymerization/depolymerization state of cytoskeletal microtubules that were encapsulated in the vesicles using pressure changes. The result indicates that it is possible to manipulate artificial cell models composed of molecules such as lipids and proteins. The findings obtained in this study will be helpful in clarifying the details of cooperation between cytoskeletal dynamics and morphogenesis of biological membranes and in improving the design and construction of further advanced artificial cell-like machinery, such as drug-delivery systems. In addition, the experimental system used in this study can be applied to research to elucidate the adaptive strategy of living organisms to external stimuli and extreme conditions such as osmotic stress and high-pressure environments like the deep sea.
• High-pressure microscopy for tracking dynamic properties of molecular machines.

  Masayoshi Nishiyama

  Biophysical chemistry ELSEVIER SCIENCE BV 231 71 - 78 0301-4622 2017/12 [Refereed]
   

  High-pressure microscopy is one of the powerful techniques to visualize the effects of hydrostatic pressures on research targets. It could be used for monitoring the pressure-induced changes in the structure and function of molecular machines in vitro and in vivo. This review focuses on the dynamic properties of the assemblies and machines, analyzed by means of high-pressure microscopy measurement. We developed a high-pressure microscope that is optimized both for the best image formation and for the stability to hydrostatic pressure up to 150 MPa. Application of pressure could change polymerization and depolymerization processes of the microtubule cytoskeleton, suggesting a modulation of the intermolecular interaction between tubulin molecules. A novel motility assay demonstrated that high hydrostatic pressure induces counterclockwise (CCW) to clockwise (CW) reversals of the Escherichia coli flagellar motor. The present techniques could be extended to study how molecular machines in complicated systems respond to mechanical stimuli.
• Reversible Morphological Control of Tubulin-Encapsulating Giant Liposomes by Hydrostatic Pressure.

  Masahito Hayashi; Masayoshi Nishiyama; Yuki Kazayama; Taro Toyota; Yoshie Harada; Kingo Takiguchi

  Langmuir : the ACS journal of surfaces and colloids AMER CHEMICAL SOC 32 (15) 3794 - 802 0743-7463 2016/04 [Refereed]
   

  Liposomes encapsulating cytoskeletons have drawn much recent attention to develop an artificial cell-like chemical-machinery; however, as far as we know, there has been no report showing isothermally reversible morphological changes of liposomes containing cytoskeletons because the sets of various regulatory factors, that is, their interacting proteins, are required to control the state of every reaction system of cytoskeletons. Here we focused on hydrostatic pressure to control the polymerization state of microtubules (MTs) within cell-sized giant liposomes (diameters ∼10 μm). MT is the cytoskeleton formed by the polymerization of tubulin, and cytoskeletal systems consisting of MTs are very dynamic and play many important roles in living cells, such as the morphogenesis of nerve cells and formation of the spindle apparatus during mitosis. Using real-time imaging with a high-pressure microscope, we examined the effects of hydrostatic pressure on the morphology of tubulin-encapsulating giant liposomes. At ambient pressure (0.1 MPa), many liposomes formed protrusions due to tubulin polymerization within them. When high pressure (60 MPa) was applied, the protrusions shrank within several tens of seconds. This process was repeatedly inducible (around three times), and after the pressure was released, the protrusions regenerated within several minutes. These deformation rates of the liposomes are close to the velocities of migrating or shape-changing living cells rather than the shortening and elongation rates of the single MTs, which have been previously measured. These results demonstrate that the elongation and shortening of protrusions of giant liposomes is repeatedly controllable by regulating the polymerization state of MTs within them by applying and releasing hydrostatic pressure.
• High-Pressure Microscopy for Controlling Microtubule Dynamics

  西山 雅祥; 瀧口 金吾; 林 真人

  顕微鏡 = Microscopy 日本顕微鏡学会 51 (2) 118 - 121 1349-0958 2016 

  細胞内にあるタンパク質は周りを水分子に取り囲まれている．筆者らは高圧力下でタンパク質と水との相互作用が変化することに着目し，タンパク質の分子構造や機能活性をコントロールしながら実時間で観察できる高圧力顕微鏡法を開発してきた．本稿では，高圧力顕微鏡の概略と細胞骨格・微小管の動態観察の結果について紹介する．
• Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium.

  Norihiro Takekawa; Masayoshi Nishiyama; Tsuyoshi Kaneseki; Tamotsu Kanai; Haruyuki Atomi; Seiji Kojima; Michio Homma

  Scientific reports NATURE PUBLISHING GROUP 5 12711 - 12711 2045-2322 2015/08 [Refereed]
   

  Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na(+) for energy coupling of the flagellar motor. The Na(+)-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.
• Direct observation of the motility of biological nanomachines at high pressure

  Masayoshi Nishiyama

  Review of High Pressure Science and Technology/Koatsuryoku No Kagaku To Gijutsu Japan Society of High Pressure Science and Technology 25 (2) 126 - 135 1348-1940 2015 [Refereed][Invited]
   

  Molecular motor is a typical molecular machinery in which the characteristic features of proteins are integrated these include enzymatic activity, energy conversion, molecular recognition and self-assembly. These biologically important reactions occur with the association of water molecules that surround the motors. Applied pressures can alter the intermolecular interactions between the motors and water. Here, we describe the development of a high-pressure microscope and a new motility assay that enables the visualization of the motility of molecular motors under conditions of high pressure.
• Viewing the rotation of molecular motors at high pressure.

  Okuno D; Nishiyama M; Noji H

  Asia Pacific Physics Newsletter 3 (2) 25 - 25 2014/08 [Invited]
  
• 力学刺激でタンパク質間相互作用を操作する:遺伝子操作も化学物質も使わない新しい生命操作技術の開発

  西山 雅祥

  化学と生物 公益社団法人 日本農芸化学会 52 (12) 782 - 784 0453-073X 2014
• Single-molecule analysis of the rotation of F₁-ATPase under high hydrostatic pressure.

  Daichi Okuno; Masayoshi Nishiyama; Hiroyuki Noji

  Biophysical journal CELL PRESS 105 (7) 1635 - 42 0006-3495 2013/10 [Refereed]
   

  F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F1 actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F1 has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F1(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å(3) and +88 Å(3) for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F1 and pressure-induced protein unfolding.
• High-pressure Microscope

  Nishiyama M; Kimura Y

  Low Temperature and Materials Sciences 京都大学低温物質科学研究センター (22) 18 - 27 1348-317X 2013/06 [Refereed][Invited]
   

  Hydrostatic pressure is one of the thermodynamic parameters that characterize the physical and chemical properties of targeted systems. Here, we report a high-pressure microscope that enables us to acquire highresolution microscopic images up to 150 MPa. The developed device allowed us to study how the pressure affects the properties in the system, e.g. bacterial motility.
• High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor.

  Masayoshi Nishiyama; Yoshiyuki Sowa; Yoshifumi Kimura; Michio Homma; Akihiko Ishijima; Masahide Terazima

  Journal of bacteriology 195 (8) 1809 - 14 0021-9193 2013/04 [Refereed]
   

  The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P.
• High Hydrostatic Pressure Induces Reversal Rotations of the Bacterial Flagellar Motor!?

  NISHIYAMA Masayoshi

  Seibutsu Butsuri The Biophysical Society of Japan General Incorporated Association 53 (5) 264 - 265 0582-4052 2013 [Refereed][Invited]
  
• Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure.

  Tomonobu M Watanabe; Katsumi Imada; Keiko Yoshizawa; Masayoshi Nishiyama; Chiaki Kato; Fumiyoshi Abe; Takamitsu J Morikawa; Miki Kinoshita; Hideaki Fujita; Toshio Yanagida

  PloS one 8 (8) e73212  2013 [Refereed]
   

  Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.
• Microscopic analysis of bacterial motility at high pressure.

  Masayoshi Nishiyama; Yoshiyuki Sowa

  Biophysical journal CELL PRESS 102 (8) 1872 - 80 0006-3495 2012/04 [Refereed]
   

  The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment.
• Bacterial motility measured by a miniature chamber for high-pressure microscopy.

  Masayoshi Nishiyama; Seiji Kojima

  International journal of molecular sciences MDPI AG 13 (7) 9225 - 39 1422-0067 2012 [Refereed]
   

  Hydrostatic pressure is one of the physical stimuli that characterize the environment of living matter. Many microorganisms thrive under high pressure and may even physically or geochemically require this extreme environmental condition. In contrast, application of pressure is detrimental to most life on Earth; especially to living organisms under ambient pressure conditions. To study the mechanism of how living things adapt to high-pressure conditions, it is necessary to monitor directly the organism of interest under various pressure conditions. Here, we report a miniature chamber for high-pressure microscopy. The chamber was equipped with a built-in separator, in which water pressure was properly transduced to that of the sample solution. The apparatus developed could apply pressure up to 150 MPa, and enabled us to acquire bright-field and epifluorescence images at various pressures and temperatures. We demonstrated that the application of pressure acted directly and reversibly on the swimming motility of Escherichia coli cells. The present technique should be applicable to a wide range of dynamic biological processes that depend on applied pressures.
• Microtubule depolymerization at high pressure

  Masayoshi Nishiyama; Yoshiki Shimoda; Manabu Hasumi; Yoshifumi Kimura; Masahide Terazima

  HIGH-PRESSURE BIOSCIENCE AND BIOTECHNOLOGY BLACKWELL PUBLISHING 1189 86 - 90 0077-8923 2010 [Refereed]
   

  We performed in vitro assays to visualize the effects of pressure on the filamentous structure of microtubules. Taxol-stabilized microtubules were tethered to kinesin motors on the observation window of a high-pressure chamber. When pressure was applied to the sample solution, all of the microtubules started to shorten from both ends. The length changes were constant over time, irrespective of the microtubule polarity. The shortening rate of microtubules increased exponentially with pressure, and the activation volume was -100 mL/mol, consistent with in vivo studies. These results show that application of pressure works directly to weaken the intermolecular interactions between tubulin molecules.
• Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

  Masayoshi Nishiyama; Yoshifumi Kimura; Yoshio Nishiyama; Masahide Terazima

  BIOPHYSICAL JOURNAL CELL PRESS 96 (3) 1142 - 1150 0006-3495 2009/02 [Refereed]
   

  Kinesin-1 is an ATP-driven molecular motor that "walks" along a microtubule by working two heads in a "hand-over-hand" fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at similar to 100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion.
• A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120

  Shigeichi Kumazaki; Makoto Hasegawa; Mohammad Ghoneim; Yugo Shimizu; Kenji Okamoto; Masayoshi Nishiyama; Hirozo Oh-Oka; Masahide Terazima

  JOURNAL OF MICROSCOPY-OXFORD BLACKWELL PUBLISHING 228 (2) 240 - 254 0022-2720 2007/11 [Refereed]
   

  We describe the construction and characterization of a laser-line-scanning microscope capable of detection of broad fluorescence spectra with a resolution of 1 nm. A near-infrared femtosecond pulse train at 800 nm was illuminated on a line (one lateral axis, denoted as X axis) in a specimen by a resonant scanning mirror oscillating at 7.9 kHz, and total multi-photon-induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron-multiplying CCD camera was used to resolve fluorescence of different colours at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. Scanning on the other two axes (Y and Z) was achieved by a closed-loop controlled sample scanning stage and a piezo-driven objective actuator. The full widths at half maximum of the point-spread function of the system were estimated to be 0.39-0.40, 0.33 and 0.56-0.59 mu m for the X (lateral axis along the line-scan), Y (the other lateral axis) and Z axes (the axial direction), respectively, at fluorescence wavelengths between 644 and 690 nm. A biological application of this microscope was demonstrated in a study of the sub-cellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena PCC7120. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of sub-cellular domains. The efficiency of the new line-scanning spectromicroscope was estimated in comparison with our own point-by-point scanning spectromicroscope. Under typical conditions of observing cyanobacterial cells, the total exposure time became shorter by about 50 times for a constant excitation density. The improvement factor was proportional to the length of the line-scanned region, as expected.
• Single molecule thermodynamics in biological motors

  Yuichi Taniguchi; Peter Karagiannis; Masayoshi Nishiyama; Yoshiharu Ishii; Toshio Yanagida

  BIOSYSTEMS ELSEVIER SCI LTD 88 (3) 283 - 292 0303-2647 2007/04 [Refereed]
   

  Biological molecular machines use thermal activation energy to carry out various functions. The process of thermal activation has the stochastic nature of output events that can be described according to the laws of thermodynamics. Recently developed single molecule detection techniques have allowed each distinct enzymatic event of single biological machines to be characterized providing clues to the underlying thermodynamics. In this study, the thermodynamic properties in the stepping movement of a biological molecular motor have been examined. A single molecule detection technique was used to measure the stepping movements at various loads and temperatures and a range of thermodynamic parameters associated with the production of each forward and backward step including free energy, enthalpy, entropy and characteristic distance were obtained. The results show that an asymmetry in entropy is a primary factor that controls the direction in which the motor will step. The investigation on single molecule thermodynamics has the potential to reveal dynamic properties underlying the mechanisms of how biological molecular machines work. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
• 生物分子モーターのステップ計測と運動解析(<シリーズ>化学から見た物性物理)

  西山 雅祥; 曽和 義幸; 石島 秋彦

  物性研究 物性研究刊行会 85 (5) 593 - 621 0525-2997 2006/02 [Refereed][Invited]
   

  この論文は国立情報学研究所の電子図書館事業により電子化されました。
• 2P240 How does high pressure affect on the bacterial motility?(39. Cell motility,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Nishiyama Masayoshi; Sowa Yoshiyuki; Kumazaki Shigeichi; Kimura Yoshifumi; Homma Michio; Ishijima Akihiko; Terazima Masahide

  Seibutsu Butsuri The Biophysical Society of Japan General Incorporated Association 46 (2) S355  2006
• Optical Tweezers

  Nishiyama M; Okamoto K

  Low Temperature and Materials Sciences 京都大学低温物質科学研究センター (7) 26 - 31 1348-317X 2005/11 [Refereed][Invited]
   

  光ピンセット（Optical Tweezers）とは、光を回折限界に集束させることで，溶液中の微粒子などをその集光点に捕捉する光技術である。この光学系を光学顕微鏡に導入することで、研究対象となる微小物体を顕微鏡で観察しながら、非接触・非侵襲で捕捉し、三次元的に自由に動かすことが可能となる。この「光の手」は、マイクロメートルスケールの物体の物性評価や生体分子の1分子計測、細胞の顕微操作など様々な分野で利用されている。本稿では、光ピンセットの原理と装置の概略を説明した後、生命科学分野での応用例について紹介したい。
• Entropy rectifies the Brownian steps of kinesin

  Y Taniguchi; M Nishiyama; Y Ishii; T Yanagida

  NATURE CHEMICAL BIOLOGY NATURE PUBLISHING GROUP 1 (6) 342 - 347 1552-4450 2005/11 [Refereed]
   

  Kinesin is a stepping motor that successively produces forward and backward 8-nm steps along microtubules. Under physiological conditions, the steps powering kinesin's motility are biased in one direction and drive various biological motile processes. The physical mechanism underlying the unidirectional bias of the kinesin steps is not fully understood. Here we explored the mechanical kinetics and thermodynamics of forward and backward kinesin steps by analyzing their temperature and load dependence. Results show that the frequency asymmetry between forward and backward steps is produced by entropy. Furthermore, the magnitude of the entropic asymmetry is 6 k(B)T, more than three times greater than expected from a current model, in which a mechanical conformational change within the kinesin molecular structure directly biases the kinesin steps forward. We propose that the stepping direction of kinesin is preferably caused by an entropy asymmetry resulting from the compatibility between the kinesin and microtubule interaction based on their polar structures.
• Single Molecule Measurements and Energetics of Biological Nanomachine

  Nishiyama Masayoshi; Harada Takahiro

  Netsu Sokutei 日本熱測定学会 32 (2) 86 - 94 0386-2615 2005 

  Movement is a fundamental characteristic of all living things. This biogenic function is attributed to molecular motors in a cell. Molecular motors are mechano-chemical enzymes that generate forces by using chemical energy derived from the hydrolysis reaction of adenosine triphosphate (ATP) molecules. Despite a large number of studies on this issue, the mechanism of mechano-chemical energy transduction is still unsolved. In this review, we describe the experimental and theoretical approaches for elucidating the mechanism how kinesin motors generate the unidirectional movement along a microtubule. By use of a novel single-molecule-detection technique, we detected the elementary processes on the sliding movement of single kinesin molecules. Motility analysis has revealed that a stochastic mechanism underlies in the unidirectional movement of kinesin. To explain the energetic aspects of the stochastic movements, we constructed a new phenomenological framework based on non-equilibrium statistical mechanics, and determined the energetic balance in single kinesin molecules. It is indicated that the hydrolysis energy of ATP is effectively used to generate the unidirectional movement. Our experimental and theoretical approaches will help to understand thermodynamics of nano-world.
• Mechano-chemical coupling of molecular motors revealed by single molecule measurements

  Y Ishii; M Nishiyama; T Yanagida

  CURRENT PROTEIN & PEPTIDE SCIENCE BENTHAM SCIENCE PUBL LTD 5 (2) 81 - 87 1389-2037 2004/04 [Refereed]
   

  Single molecule measurements have allowed series of kinetic events of biomolecules to be monitored without interruption. The stepwise movement Of molecular motors was measured and analyzed in relation to the hydrolysis reaction of ATP. In the case of kinesin, forward and backward Steps Occurred stochastically at the same chemical state. The directional movement was explained by the asymmetric potential created by the interaction between kinesin and microtubules. Similarly thermal Brownian movement of myosin during the hydrolysis of single ATP molecules was biased through an asymmetric potential, resulting in directional movement. Thus, single molecule measurements have provided new approaches to analyze the function of molecular motors which often consist of several different events.
• Feynman's Thermal Ratchet Mechanism on Kinesin Motors

  NISHIYAMA Masayoshi; HIGUCHI Hideo

  Biophysics 一般社団法人日本生物物理学会 44 (2) 75 - 80 0582-4052 2004/03 [Refereed][Invited]
   

  Kinesin is an ATP-driven molecular motor that moves processively along a microtubule in a stepwise manner. The steps occur not only in the forward direction, but also in the backward. Here, we have studied the bidirectional stepping mechanism of kinesin motors. The stepping mechanism of the forward and backward movements was well characterized by Feynman's thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by utilizing the free energy released from the hydrolysis of ATP.
• Single molecule processes on the stepwise movement of ATP-driven molecular motors

  M Nishiyama; H Higuchi; Y Ishii; Y Taniguchi; T Yanagida

  BIOSYSTEMS ELSEVIER SCI LTD 71 (1-2) 145 - 156 0303-2647 2003/09 [Refereed]
   

  Movement is a fundamental characteristic of all living things. This biogenic function that is attributed to the molecular motors such as kinesin, dynein and myosin. Molecular motors generate forces by using chemical energy derived from the hydrolysis reaction of ATP molecules. Despite a large number of studies on this topic, the chemomechanical energy transduction mechanism is still unsolved. In this study, we have investigated the chemomechanical coupling of the ATPase cycle to the mechanical events of the molecular motor kinesin using single molecule detection (SMD) techniques. The SMD techniques allowed to detection of the movement of single kinesin molecules along a microtubule and showed that kinesin steps mainly in the forward direction, but occasionally in the backward. The stepping direction is determined by a certain load-dependent process, on which the stochastic behavior is well characterized by Feynman's thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by using the free energy released from the hydrolysis of ATP. (C) 2003 Elsevier Ireland Ltd. All rights reserved.
• Chemomechanical coupling of the forward and backward steps of single kinesin molecules

  M Nishiyama; H Higuchi; T Yanagida

  NATURE CELL BIOLOGY NATURE PUBLISHING GROUP 4 (10) 790 - 797 1465-7392 2002/10 [Refereed]
   

  The molecular motor kinesin travels processively along a microtubule in a stepwise manner. Here we have studied the chemomechanical coupling of the hydrolysis of ATP to the mechanical work of kinesin by analysing the individual stepwise movements according to the directionality of the movements. Kinesin molecules move primarily in the forward direction and only occasionally in the backward direction. The hydrolysis of a single ATP molecule is coupled to either the forward or the backward movement. This bidirectional movement is well described by a model of Brownian motion assuming an asymmetric potential of activation energy. Thus, the stepwise movement along the microtubule is most probably due to Brownian motion that is biased towards the forward direction by chemical energy stored in ATP molecules.
• Single Molecular Measurement and Manipulation by Optical tweezers

  NISHIYAMA Masayoshi

  Japanese Journal of Optics,30, 445-450 応用物理学会分科会日本光学会 30 (7) 445 - 450 0389-6625 2001/07 [Refereed]
  
• Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules

  M Nishiyama; E Muto; Y Inoue; T Yanagida; H Higuchi

  NATURE CELL BIOLOGY MACMILLAN PUBLISHERS LTD 3 (4) 425 - 428 1465-7392 2001/04 [Refereed]
   

  Kinesin is a molecular motor that moves processively(1-4) by regular 8-nm steps along microtubules(5-11). The processivity of this movement is explained by a hand-overhand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alpha beta -tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle(9-13). To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of similar to4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head(14). It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.

Books etc

• 極限環境微生物の先端科学と社会実装最前線

  西山, 雅祥; 八木, 俊樹 (Contributor1-5-5 極限環境で復活する鞭毛運動)エヌ・ティー・エス 2023/12 9784860438487 3, 12, 469, 12p
• 高圧力の科学・技術辞典

  西山 雅祥 (Contributor高圧力顕微鏡による細胞運動観察)朝倉書店 2022
• The Role of Water in ATP Hydrolysis Energy Transduction

  Masayoshi NISHIYAMA (ContributorChapter 19: Controlling motility of ATP-driven molecular motors with high hydrostatic pressure, p.325-337)Springer 2018/05
• 高度物理刺激と生体応答

  西山 雅祥 (Contributor高圧力顕微鏡法によるタンパク質分子機械の回転制御, p.59-62)養賢堂 2017/08
• The bacterial flagellum : methods and protocols

  Minamino, Tohru; Namba, Keiichi (ContributorChapter 13: Tracking the movement of a single prokaryotic cell at extreme environmental conditions.)Humana Press 2017/04 9781493969265 337
• HIgh Pressure Bioscience – Basic Concepts, Applications and Frontiers

  NISHIYAMA Masayoshi (ContributorChapter 27, High-pressure microscopy for studying molecular motors, p.593-611)Springer 2015/07
• High Pressure Bioscience and Biotechnology

  Nishiyama Masayoshi (ContributorBacterial Motility Measured by High-Pressure Microscopy)さんえい出版 2015
• Single Molecule Detection in Solution

  Masayoshi NISHIYAMA (ContributorStudying Molecular Motors on the Single Molecule Level, p.273-292)WILEY-VCH 2002

Conference Activities & Talks

• 高圧刺激下での細胞動態の可視化

  森松賢順; 西山雅祥; 成瀬恵治

  日本生体医工学会大会プログラム・抄録集(Web)  2022
• Mechanism of molecular machines  [Invited]

  Masayoshi NISHIYAMA

  Across nonlinear and nonequilibrium physics in memory of Dr. Takahiro Harada  2021/03
• Intracellular Calcium Concentration-independent Cardiomyocyte Contraction Triggered by High Hydrostatic Pressure

  Yohei Yamaguchi; Masayoshi Nishiyama; Gentaro Iribe; Keiji Naruse; Masatoshi Morimatsu

  Biophysical Journal  2021/02  Elsevier BV
  
• Optical Manipulation at Different Pressures  [Invited]

  Masayoshi NISHIYAMA

  The 58th Annual Meeting of the Biophysical Society of Japan  2020/09
• 高圧力下で活性化される生命をみる  [Invited]

  西山 雅祥

  理工学総合研究所コロキウム  2020/09
• Vigorous flagellar beating in Chlamydomonas axonemes at high-pressure

  八木俊樹; 西山雅祥

  応用物理学会春季学術講演会講演予稿集(CD-ROM)  2020
• Vigorous flagellar beating in Chlamydomonas axonemes at high-pressure

  八木俊樹; 西山雅祥

  応用物理学会秋季学術講演会講演予稿集(CD-ROM)  2020
• Activation of the motility machineries using high-pressure techniques.  [Invited]

  Masayoshi NISHIYAMA

  第57回日本生物物理学会年会  2019/09
• 静水圧を力学刺激とする生命操作技術の開発  [Invited]

  西山 雅祥

  日本メカノバイオロジー研究会  2019/09
• 力刺激による生命活動の活性化コントロール  [Invited]

  西山 雅祥

  名古屋大学理学部セミナー  2019/02
• 周期的伸展刺激と静水圧刺激に対するヒト歯根膜細胞の形態と配向の変化

  藤田彩乃; 森松賢順; 西山雅祥; 高柴正悟; 成瀬恵治

  日本生体医工学会大会プログラム・抄録集(Web)  2019
• 生理的圧力下での細胞動態計測

  森松賢順; 藤田彩乃; 綾晃記; 寺町一希; 西山雅祥; 成瀬恵治

  日本生体医工学会大会プログラム・抄録集(Web)  2019
• Escherichia coli由来新規リゾホスファチジン酸アシル基転移酵素YihGの基質特異性と生理機能解析

  豊竹洋佑; 川本純; 西山雅祥; 小川拓哉; 栗原達夫

  日本農芸化学会大会講演要旨集(Web)  2019
• 生理的高圧下での細胞動態計測

  森松賢順; 西山雅祥; 成瀬恵治

  日本分子生物学会年会プログラム・要旨集(Web)  2019
• 高静水圧下においてクラミドモナス非運動性変異株に誘導される繊毛運動

  八木俊樹; 西山雅祥

  日本細胞生物学会大会(Web)  2019
• 歯根膜細胞における機械刺激による恒常性への影響

  藤田 彩乃; 森松 賢順; 西山 雅祥; 成瀬 恵治; 高柴 正悟

  日本歯周病学会会誌  2018/10  (NPO)日本歯周病学会
  
• Pressure-induced activation of the cell motility  [Invited]

  西山 雅祥

  第56回日本生物物理学会年会  2018/09
• Effects of Mechanical Stress on Periodontal Ligament  [Not invited]

  Fujita Ayano; Morimatsu Masatoshi; Nishiyama Masayoshi; Takashiba Shogo; Naruse Keiji

  BIOPHYSICAL JOURNAL  2018/02
• Pressure-Ineduced Activation of the Swimming Motility of Magnetotactic Bacterium

  Masayoshi Nishiyama; Ruan Juanfang; Takayuki Kato; Toru Minamino; Keiichi Namba; Akitoshi Seiyama; Long-Fei Wu; Yoshie Harada

  Biophysical Journal  2018/02  Elsevier BV
  
• 歯根膜細胞における機械刺激による恒常性への影響

  藤田彩乃; 森松賢順; 西山雅祥; 成瀬恵治; 高柴正悟

  日本歯周病学会会誌(Web)  2018
• 歯根膜細胞における機械刺激による恒常性への影響

  藤田彩乃; 森松賢順; 西山雅祥; 高柴正悟; 成瀬恵治

  高圧討論会講演要旨集  2018
• シェアストレスによる血管内皮細胞の負荷応答の解析

  稲葉晃帆; 成瀬恵治; 森松賢順; 藤田彩乃; 西山雅祥

  高圧討論会講演要旨集  2018
• 新規リゾホスファチジン酸アシル基転移酵素YihGのEscherichia coli膜リン脂質生合成への寄与とその生理機能

  豊竹洋佑; 川本純; 西山雅祥; 小川拓哉; 栗原達夫

  日本生化学会大会(Web)  2018
• カスケード型超並列シミュレーションで見るタンパク質間結合の圧力依存性

  畑宏明; 西原泰孝; 西山雅祥; 川岸郁朗; 北尾彰朗

  日本蛋白質科学会年会プログラム・要旨集  2018
• 高圧下での細胞動態イメージング

  森松賢順; 藤田彩乃; 綾晃記; 西山雅祥; 成瀬恵治

  日本生体医工学会大会プログラム・抄録集(Web)  2018
• 生理的高圧下でのリアルタイム細胞動態計測

  森松賢順; 藤田彩乃; 綾晃記; 寺町一希; 稲葉晃帆; 成瀬恵治; 西山雅祥

  高圧討論会講演要旨集  2018
• 線虫C.elegansのDAF-16/FoxOを介した新たな静水圧応答

  渡辺尚; 渡辺尚; 森松賢順; 西山雅祥; 渡邉賢; 鍵裕之; 成瀬恵治; 東谷篤志

  日本放射線影響学会大会抄録(Web)  2018
• 静水圧負荷による軟骨細胞のシグナル伝達機構の解明

  寺町一希; 成瀬恵治; 森松賢順; 藤田彩乃; 西山雅祥

  高圧討論会講演要旨集  2018
• Pressure-induced activation of the swimming motility of magnetotactic bacterium.  [Not invited]

  Nishiyama, M; Juanfang, R; Kato, T; Minamino, T; Namba, K; Seiyama, A; Long-Fei Wu; Harada, Y

  Biophysical. Journal  2017/11
• 静水圧を用いた細胞活動の活性化イメージング  [Invited]

  西山 雅祥

  同志社大学 セミナー  2017/10
• 高圧力顕微鏡法による細胞活動の活性化イメージング  [Invited]

  西山 雅祥

  東北大学工学部セミナー  2017/03
• Pressure Effects on Dissociation of CheY-FliM Complex Studied by Molecular Dynamics Simulations  [Not invited]

  Hata Hiroaki; Nishihara Yasutaka; Nishiyama Masayoshi; Kawagishi Ikuro; Kitao Akio

  BIOPHYSICAL JOURNAL  2017/02
• Reversible Morphological Control of Tubulin-Encapsulating Giant Liposomes by Hydrostatic Pressure  [Not invited]

  Masayoshi Nishiyama; Masahito Hayashi; Kingo Takiguchi; Yoshie Harada

  BIOPHYSICAL JOURNAL  2017/02  CELL PRESS
  
• 高圧力顕微鏡法を用いたヒト由来歯根膜細胞の動態イメージング

  藤田彩乃; 森松賢順; 西山雅祥; 高柴正悟; 成瀬恵治

  高圧討論会講演要旨集  2017
• ヒト由来歯根膜細胞の力学刺激応答イメージング

  藤田彩乃; 森松賢順; 西山雅祥; 高柴正悟; 成瀬恵治

  応用物理学会秋季学術講演会講演予稿集(CD-ROM)  2017
• 生きた細胞内で働く分子機械を高圧力顕微鏡法でみる  [Invited]

  西山 雅祥

  第39回 日本分子生物学会年会  2016/12
• 高静水圧環境の分子ダイナミクスを直接みる  [Invited]

  西山 雅祥

  第59回日本顕微鏡学会シンポジウム  2016/11
• 高圧力環境にある生命活動を直接観察する  [Invited]

  西山 雅祥

  麻酔メカニズム研究会歓談会  2016/10
• 3SFA-04 High-pressure microscopy for manipulating cellular architecture and function  [Invited]

  NISHIYAMA Masayoshi

  Annual Meeting of the Biophysical Society of Japan  2016/09
• 高圧力顕微鏡を用いた深海微生物の運動観察  [Invited]

  西山 雅祥

  第6回分子モーター討論会  2016/07
• 高圧力顕微鏡を使った生体運動イメージング  [Invited]

  西山 雅祥

  Bio-Divセミナー, JAMSTEC  2016/04
• 高圧力顕微鏡法による細胞構造コントロール  [Invited]

  西山 雅祥

  第55回日本生体医工学会大会  2016/04
• Sodium-Driven Energy Conversion for Flagellar Rotation of the Earliest Divergent Hyperthermophilic Bacterium  [Not invited]

  Norihiko Takekawa; Masayoshi Nishiyama; Tsuyoshi Kaneseki; Tamotsu Kanai; Haruyuki Atomi; Seij Kojima; Michio Homma

  BIOPHYSICAL JOURNAL  2016/02  CELL PRESS
  
• 高圧力顕微鏡を用いた細菌の走化性応答コントロール

  西山雅祥; 沢田孝; 曽和義幸; 原田慶恵; 川岸郁朗

  高圧討論会講演要旨集  2016
• 圧力応答を示すYFP挿入変異体の高圧下での構造

  辻井美香; 永江峰幸; 田中るみか; 慶澤景子; 渡邊朋信; 西山雅祥; 渡邊信久; 川口辰也; 今田勝巳

  日本蛋白質科学会年会プログラム・要旨集  2016
• 圧力変化を利用した細胞骨格封入膜小胞の形態制御

  瀧口金吾; 林真人; 風山祐輝; 豊田太郎; 原田慶恵; 西山雅祥

  生体膜と薬物の相互作用シンポジウム講演要旨集  2016
• 高圧力顕微鏡法による細菌べん毛の構造変化イメージング

  西山雅祥; 曽和義幸

  応用物理学会春季学術講演会講演予稿集(CD-ROM)  2016
• 高圧力顕微鏡法によるタンパク質分子間相互作用コントロール

  西山雅祥; 林真人; 瀧口金吾; 原田慶恵

  応用物理学会秋季学術講演会講演予稿集(CD-ROM)  2016
• タンパク質分子機械の力学変調イメージング  [Invited]

  西山 雅祥

  北海道大学フロンティア化学教育研究センター  2015/08
• 超好熱菌Aquifex aeolicusの運動解析と大腸菌を用いたAquifexモーター固定子の機能解析

  本間 道夫; 小嶋 誠司; 竹川 宜宏; 西山 雅祥; 金関 剛史; 金井 保

  日本細菌学雑誌  2015/02  日本細菌学会
  
• 深海性Shewanella属細菌由来シトクロムcの圧力適応機構

  政成美沙; 西山雅祥; 三本木至宏

  日本農芸化学会大会講演要旨集(Web)  2015
• クラミドモナス非運動性変異株の力学コントロール

  八木俊樹; 西山雅祥

  応用物理学会春季学術講演会講演予稿集(CD-ROM)  2015
• Bacterial Motility Measured by a Miniature Chamber for High-Pressure Microscopy  [Not invited]

  Masayoshi Nishiyama; Seiji Kojima

  BIOPHYSICAL JOURNAL  2015/01  CELL PRESS
  
• 力学刺激で細胞運動を操作する  [Invited]

  西山 雅祥

  第103回生命機能研究科コロキウム  2014/12
• 高圧力を用いたタンパク質分子機械の運動変調イメージング  [Invited]

  西山 雅祥

  第55回高圧討論会  2014/11
• Single molecule analysis of ATP-driven molecular motors at high pressure  [Invited]

  西山 雅祥

  7th International Meeting on Biomolecules under Pressure  2014/07
• 超好熱菌Aquifex aeolicusのべん毛運動とモーター固定子タンパク質の機能解析・精製

  竹川宜宏; 西山雅祥; 土方敦司; 金関剛史; 郷原瑞樹; 真柳浩太; 小嶋誠司; 金井保; 白井剛; 本間道夫

  日本生体エネルギー研究会討論会講演要旨集  2014
• 高圧力顕微鏡の開発とバクテリア・べん毛モーターの運動変調

  西山雅祥; 曽和義幸

  応用物理学会春季学術講演会講演予稿集(CD-ROM)  2014
• 1P214 Reversible morphological control of tubulin-encapsulated giant-liposomes induced by change of hydrostatic pressure and temperature(13B. Biological & Artificial membrane: Dynamics,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

  Hayashi Masahito; Nishiyama Masayoshi; Kazayama Yuki; Toyota Taro; Takiguchi Kingo

  Seibutsu Butsuri  2014  The Biophysical Society of Japan General Incorporated Association
  
• 2P175 High pressure induces flagellar bending movements in Chlamydomonas paralyzed mutants(12. Cell biology,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

  Yagi Toshiki; Nishiyama Masayoshi

  Seibutsu Butsuri  2014  The Biophysical Society of Japan General Incorporated Association
  
• Pressure-Speed Relationship of the Sodium-Driven Flagellar Motor of Vibrio Alginolyticus  [Not invited]

  Masayoshi Nishiyama; Yoshiki Shimoda; Yoshifumi Kimura; Masahide Terazima; Michio Homma; Seiji Kojima

  BIOPHYSICAL JOURNAL  2014/01  CELL PRESS
  
• Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure  [Not invited]

  Daichi Okuno; Masayoshi Nishiyama; Hiroyuki Noji

  BIOPHYSICAL JOURNAL  2014/01  CELL PRESS
  
• 2SDP-02 Controlling the molecular machinery by water molecules of the hydration(Biomolecular machinery driven by surrounding water,Symposium,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))  [Not invited]

  Nishiyama Masayoshi

  Seibutsu Butsuri  2014  The Biophysical Society of Japan General Incorporated Association
  
• 高圧力を用いたタンパク質分子機械の構造変化イメージング  [Invited]

  西山 雅祥

  日本機械学会2013年度年次大会  2013/09
• W271003 Visualization of the Structural Change of Molecular Machines at High Pressure

  NISHIYAMA Masayoshi

  Mechanical Engineering Congress, Japan  2013/09  The Japan Society of Mechanical Engineers
   

  The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20℃, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P.
• Shewanella violacea由来cytochrome c₅の圧力耐性

  政成美沙; 若井暁; 加藤千明; 西山雅祥; 三本木至宏

  日本農芸化学会関西支部講演会講演要旨集  2013
• Microscopic analysis of bacterial motility at high pressure  [Invited]

  NISHIYAMA Masayoshi

  9th International Conference on Flow Dynamics  2012/09
• Single molecule analysis of ATP-driven molecular motors at high pressure  [Invited]

  NISHIYAMA Masayoshi

  7th International Meeting on Biomolecules under Pressure  2012/09
• 8C34 Pressure-induced modulation on the motility of bio-nanomachines.  [Invited]

  NISHIYAMA Masayoshi

  バイオエンジニアリング講演会講演論文集  2012/01
• 高圧力に耐える深海微生物のべん毛運動

  仲宗根薫; 山野由美子; 加藤千明; 西山雅祥

  日本農芸化学会大会講演要旨集(Web)  2012
• 3H0924 Resurrection of flagellar bending movements in chlamydomonas paralyzed mutants at high pressure(Cell Biology III:Cytoskeleton & Motility,Oral Presentation)

  Yagi Toshiki; Nishiyama Masayoshi

  Seibutsu Butsuri  2012  The Biophysical Society of Japan General Incorporated Association
  
• Pressure Sensitive Reaction of F1-ATPase  [Not invited]

  Daichi Okuno; Masayoshi Nishiyama; Hiroyuki Noji

  BIOPHYSICAL JOURNAL  2012/01  CELL PRESS
  
• Intracellular Pressure Measurement by using Pressure Sensitive Yellow Fluorescent Protein  [Not invited]

  Tomonobu M. Watanabe; Katsumi Imada; Keiko Yoshizawa; Masayoshi Nishiyama; Chiaki Kato; Fumiyoshi Abe; Toshio Yanagida

  BIOPHYSICAL JOURNAL  2012/01  CELL PRESS
  
• Enhancing Temperature and Pressure Sensitivity of Various Fluorescent Proteins  [Not invited]

  Takamitsu Morikawa; Masayoshi Nishiyama; Keiko Yoshizawa; Toshio Yanagida; Tomonobu Watanabe

  BIOPHYSICAL JOURNAL  2012/01  CELL PRESS
  
• Microscopic Analysis of Bacterial Motility at High Pressure  [Not invited]

  Masayoshi Nishiyama; Yoshiyuki Sowa

  BIOPHYSICAL JOURNAL  2012/01  CELL PRESS
  
• 2SK-01 High-pressure microscopy reveals the mechanism how applied pressures affect on the bacterial motility(2SK Pressure opens new paradigm of bioscience,The 49th Annual Meeting of the Biophysical Society of Japan)  [Invited]

  Nishiyama Masayoshi

  Biophysics  2011/08
• Thermodyanmic Control on the Torque Generation of Bacterial Flagellar Motors  [Not invited]

  Manabu Hasumi; Masahide Terazima; Masayoshi Nishiyama

  BIOPHYSICAL JOURNAL  2011/02  CELL PRESS
  
• F₁-ATPaseの圧力を変調手段とした一分子回転観察

  奥野大地; 西山雅祥; 野地博行; 野地博行

  日本生体エネルギー研究会討論会講演要旨集  2011
• 超好熱始原菌の遊泳運動観察

  西山雅祥; 西山雅祥; 塚本遼平; 吉川雅英; 今中忠行; 金井保

  日本Archaea研究会講演会要旨集  2010
• ロータリー型分子モーターのトルク発生コントロールと運動計測

  西山雅祥; 西山雅祥

  日本生体エネルギー研究会討論会講演要旨集  2010
• 3P153 Effect of pressure on the tprque-speed relationship of bacterial illagellar motor(Molecular motor,The 48th Annual Meeting of the Biophysical Society of Japan)

  Hasumi Manabu; Terazima Masahide; Nishiyama Masayoshi

  Seibutsu Butsuri  2010  The Biophysical Society of Japan General Incorporated Association
  
• High-Pressure Microscopy for Modulating the Structure and Function of Biomolecules  [Not invited]

  Masayoshi Nishiyama; Yoshifumi Kimura; Masahide Terazima

  BIOPHYSICAL JOURNAL  2010/01  CELL PRESS
  
• 1YP1-05 Thermodynamic control of bacterial flagellar motors(1YP1 Early Research in Biophysics Award Candidate Presentations,The 47th Annual Meeting of the Biophysical Society of Japan)  [Invited]

  Nishiyama Masayoshi; Sowa Yoshiyuki; Kumazaki Shigeichi; Kimura Yoshifumi; Homma Michio; Ishijima Akihiko; Terazima Masahide

  Biophysics  2009/09
• 1P-126 Thermodynamic control of bacterial flagellar motors(Molecular motor, The 47th Annual Meeting of the Biophysical Society of Japan)

  Nishiyama Masayoshi; Sowa Yoshiyuki; Kumazaki Shigeichi; Kimura Yoshifumi; Homma Michio; Ishijima Akihiko; Terazima Masahide

  Seibutsu Butsuri  2009  The Biophysical Society of Japan General Incorporated Association
  
• 2S3-4 Modulation of the flagellar motor rotation by high-pressure microscopy(2S3 Structure and functional mechanism of the bacterial flagellar motor,The 46th Annual Meeting of the Biophysical Society of Japan)  [Invited]

  Nishiyama Masayoshi

  Biophysics  2008/10
• 高圧力顕微鏡の開発と生体分子間相互作用変調イメージング

  西山雅祥; 西山雅祥

  高圧討論会講演要旨集  2008
• 3P-153 Development of a high-pressure microscope for measuring nanometer-displacements of single molecular motors(The 46th Annual Meeting of the Biophysical Society of Japan)

  Hasumi Manabu; Kimura Yoshifumi; Terazima Masahide; Nishiyama Masayoshi

  Seibutsu Butsuri  2008  The Biophysical Society of Japan General Incorporated Association
  
• 1P-170 Pressure-induced effects of the motility of Vibrio alginolyticus(The 46th Annual Meeting of the Biophysical Society of Japan)

  Shimoda Yoshiki; Nishiyama Masayoshi; Kojima Seiji; Homma Michio; Kimura Yoshifumi; Terazima Masahide

  Seibutsu Butsuri  2008  The Biophysical Society of Japan General Incorporated Association
  
• 3P-328 High-pressure microscopy for single molecule imaging and nano-manipulation(The 46th Annual Meeting of the Biophysical Society of Japan)

  Nishiyama Masayoshi; Kimura Yoshifumi; Terazima Masahide

  Seibutsu Butsuri  2008  The Biophysical Society of Japan General Incorporated Association
  
• 2P328 A Line-Scan Multiphoton Broad-Fluorescence Spectromicroscope Applied to the Thylakoid Membrane of a Cyanobacterium Anabaena(Photobiology-photosynthesis,Poster Presentations)  [Not invited]

  Kumazaki Shigeichi; Hasegawa Makoto; Ghoneim Mohammad; Shimizu Yugo; Okamoto Kenji; Nishiyama Masayoshi; Oh-oka Hirozo; Terazima Masahide

  Biophysics  2007/11  The Biophysical Society of Japan
  
• 高圧力によるべん毛モーターの逆回転運動(Pressure-induced reversal in the rotational direction of the bacterial flagellar motor)

  西山 雅祥; 曽和 義幸; 熊崎 茂一; 木村 佳文; 本間 道夫; 石島 秋彦; 寺嶋 正秀

  生物物理  2007/11  (一社)日本生物物理学会
  
• Na+駆動型べん毛モーターにおける遊泳速度の圧力応答(Pressure-velocity relationship of sodium-driven flagellar motor of Vibrio alginolyticus)

  下田 義樹; 西山 雅祥; 小嶋 誠司; 本間 道夫; 木村 佳文; 寺嶋 正秀

  生物物理  2007/11  (一社)日本生物物理学会
  
• 3次元顕微分光法による植物葉緑体とシアノバクテリア中の光合成膜の環境順応の観察  [Not invited]

  熊崎茂一; 長谷川慎; GHONEIM Mohammad; 西山雅祥; 寺嶋正秀; 岡本憲二; 大岡宏造; 椎名隆

  日本化学会講演予稿集  2007/03
• 3P176 Pressure-induced reversal in the rotational direction of the bacterial flagellar motor(Molecular motors,Oral Presentations)

  Nishiyama Masayoshi; Sowa Yoshiyuki; Kumazaki Shigeichi; Kimura Yoshifumi; Homma Michio; Ishijima Akihiko; Terazima Masahide

  Seibutsu Butsuri  2007  The Biophysical Society of Japan General Incorporated Association
  
• 3P177 Pressure-velocity relationship of sodium-driven flagellar motor of Vibrio alginolyticus(Molecular motors,Oral Presentations)

  Shimoda Yoshiki; Nishiyama Masayoshi; Kojima Seiji; Homma Michio; Kimura Yoshihumi; Terazima Masahide

  Seibutsu Butsuri  2007  The Biophysical Society of Japan General Incorporated Association
  
• Microscopic analysis of kinesin-microtubule complex under high hydrostatic pressure  [Not invited]

  Masayoshi Nishiyama; Yoshifumi Kimura; Masahide Terazima

  BIOPHYSICAL JOURNAL  2007/01  BIOPHYSICAL SOCIETY
  
• 高時間分解で状態遷移軌跡を復元する単一分子蛍光計測法  [Not invited]

  岡本憲二; 西山雅祥; 熊崎茂一; 寺嶋正秀

  応用物理学会学術講演会講演予稿集  2006/08
• cytochrome c分子の構造揺らぎの実時間単一分子蛍光計測  [Not invited]

  岡本憲二; 西山雅祥; 寺嶋正秀

  分子構造総合討論会講演要旨集(CD−ROM)  2006/08
• 単一タンパク質分子の構造変化ダイナミクスの実時間観察  [Not invited]

  岡本憲二; 西山雅祥; 寺嶋正秀

  応用物理学関係連合講演会講演予稿集  2006/03
• 植物型光合成膜の環境順応変化を捉える顕微分光法の実践と改良

  熊崎茂一; MOHAMMAD Ghoneim; 長谷川慎; 西山雅祥; 清水祐吾; 寺嶋正秀; 大岡宏造

  分子構造総合討論会講演要旨集(CD-ROM)  2006
• Adapatation dynamics of chloroplast fine structure studied by misroscopic fluorescence spectroimages

  熊崎茂一; 長谷川慎; 谷口太郎; GHONEIM Mohamed; 池上勇; 西山雅祥; 寺嶋正秀; 大岡宏造

  日本化学会講演予稿集  2006
• 高静水圧下でのタンパク質フィラメントの顕微観測

  西山雅祥; 木村佳文; 寺嶋正秀

  高圧討論会講演要旨集  2006
• 1P544 Direct observation of unfolding-refolding dynamics of single cytochrome c proteins with milliseconds time resolution(26. Single molecule biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Okamoto Kenji; Nishiyama Masayoshi; Terazima Masahide

  Seibutsu Butsuri  2006  The Biophysical Society of Japan General Incorporated Association
  
• 2P240 How does high pressure affect on the bacterial motility?(39. Cell motility,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Nishiyama Masayoshi; Sowa Yoshiyuki; Kumazaki Shigeichi; Kimura Yoshifumi; Homma Michio; Ishijima Akihiko; Terazima Masahide

  Seibutsu Butsuri  2006  The Biophysical Society of Japan General Incorporated Association
  
• 植物型光合成を担う光合成膜の3次元顕微画像と顕微分光

  熊崎茂一; 長谷川慎; 谷口太郎; GHONEIM Mohammad; 池上勇; 西山雅祥; 大岡宏造; 寺嶋正秀

  光化学討論会講演要旨集  2005
• 高圧蛍光顕微鏡の開発と生体分子への応用

  西山雅祥; 西山嘉男; 寺嶋正秀; 木村佳文

  高圧討論会講演要旨集  2005
• 2P197 Kinetic mechanism of the kinesin steps

  Taniguchi Y.; Nishiyama M.; Ishii Y.; Yanagida T.

  Seibutsu Butsuri  2005  The Biophysical Society of Japan General Incorporated Association
  
• 2P032 Microscopic analysis of kinesin-microtubule cmplex under high pressure

  Nishiyama M.; Kimura Y.; Nishiyama Y.; Terazima M.

  Seibutsu Butsuri  2005  The Biophysical Society of Japan General Incorporated Association
  
• Steppping kinetics of kinesin motors is described by load-independent Michaelis-Menten constant  [Not invited]

  M Nishiyama; Y Ishii; T Yanagida; M Terazima

  BIOPHYSICAL JOURNAL  2005/01  BIOPHYSICAL SOCIETY
  
• 3P186 Steric entropy barriers directing Brownian stepping movement of kinesin

  Taniguchi Y.; Nishiyama M.; Ishii Y.; Yanagida T.

  Seibutsu Butsuri  2004  The Biophysical Society of Japan General Incorporated Association
  
• 2P286 Development of a high-pressure fluorescent microscope

  Nishiyama M.; Kimura Y.; Terazima M.

  Seibutsu Butsuri  2004  The Biophysical Society of Japan General Incorporated Association
  
• Forward and backward movements of single kinesin molecules described by free energy landscape  [Not invited]

  Y Taniguchi; M Nishiyama; Y Ishii; T Yanagida

  BIOPHYSICAL JOURNAL  2004/01  BIOPHYSICAL SOCIETY
  
• 生体分子モーターの1分子計測と動作メカニズム

  西山雅祥; 寺嶋正秀

  分子構造総合討論会講演要旨集(CD-ROM)  2003
• ファインマンのサーマルラチェットモデルによるキネシンの運動解析

  西山雅祥; 樋口秀男; 柳田敏雄

  日本生物物理学会年会講演予稿集  2002
• キネシンのステップ生成過程の温度依存性

  谷口雄一; 西山雅祥; 石井由晴; 柳田敏雄

  日本生物物理学会年会講演予稿集  2002
• 生体分子モーターの1分子計測と運動解析

  西山雅祥; 武藤悦子; 井上裕一; 柳田敏雄; 樋口秀男

  光化学討論会講演要旨集  2002
• 1O1515 Temperature dependence of the stepping movement of single kinesin molecules

  Taniguchi Y.; Nishiyama M.; Ishii Y.; Yanagida T.

  Seibutsu Butsuri  2002  The Biophysical Society of Japan General Incorporated Association
  
• 1O1545 Motility analysis of single kinesin molecules by Feynman's thermal ratchet model.

  Nishiyama M.; Higuchi H.; Yanagida T.

  Seibutsu Butsuri  2002  The Biophysical Society of Japan General Incorporated Association
  
• Stepping kinetics of the bidirectional movements of single kinesin molecules  [Not invited]

  M Nishiyama; T Yanagida; H Higuchi

  BIOPHYSICAL JOURNAL  2002/01  BIOPHYSICAL SOCIETY
  
• Rapid substeps detected in the 8-nm step of the kinesin walking cycle  [Not invited]

  H Higuchi; M Nishiyama

  BIOPHYSICAL JOURNAL  2002/01  BIOPHYSICAL SOCIETY
  
• 力生成過程におけるキネシンの確率過程運動モデル

  西山雅祥; 柳田敏雄; 樋口秀男

  日本生物物理学会年会講演予稿集  2001
• 生物分子モーターの1分子力学計測

  西山雅祥; 柳田敏雄; 柳田敏雄; 樋口秀男

  応用物理学会学術講演会講演予稿集  2001
• Biased Brownian motion model for the force generation of kinesin

  Nishiyama M.; Yanagida T.; Higuchi H.

  Seibutsu Butsuri  2001  The Biophysical Society of Japan General Incorporated Association
  
• Load dependence of the stepping rate for single kinesin molecules  [Not invited]

  M Nishiyama; T Yanagida; H Higuchi

  BIOPHYSICAL JOURNAL  2001/01  BIOPHYSICAL SOCIETY
  
• キネシン1分子の運動を光ピンセットで探る

  西山雅祥; 武藤悦子; 井上裕一; 柳田敏雄; 樋口秀男

  日本分子生物学会年会プログラム・講演要旨集  2000
• 光捕捉を用いた生体分子モーターの1分子力学計測(II)

  西山雅祥; 武藤悦子; 井上裕一; 柳田敏雄; 柳田敏雄; 樋口秀男

  応用物理学会学術講演会講演予稿集  2000
• Sub-steps within the 8nm step per ATP cycle of kinesin molecules

  Nishiyama M.; Muto E.; Inoue Y.; Yanagida T.; H. Hideo

  Seibutsu Butsuri  2000  The Biophysical Society of Japan General Incorporated Association
  
• キネシン1分子の高時間分解能計測

  西山雅祥; 樋口秀男; 武藤悦子; 井上裕一; 柳田敏雄

  日本生物物理学会年会講演予稿集  1999
• 光捕捉を用いた分子モーターの1分子力学測定

  西山雅祥; 樋口秀男; 武藤悦子; 井上裕一; 柳田敏雄

  応用物理学会学術講演会講演予稿集  1999
• Detection of nmdisplacement of single kinesin molecutes at high temporal resolution

  Nishiyama M.; Hideo H.; Muto E.; Inoue Y.; Yanagida T.

  Seibutsu Butsuri  1999  The Biophysical Society of Japan General Incorporated Association
  
• Derection of sub-8nm steps of single kinesin molecules at high-temporal resolution

  Nishiyama M.; H. Hideo; Muto E.; Inoue Y.; Yanagida T.

  Biophysics  1998/09  The Biophysical Society of Japan General Incorporated Association
  
• Laser trapping nanometry and imaging of single kinesin molecules moving along microtubules.

  Nonoyama Y.; Inoue Y.; Nishiyama M.; Miyai T.; Shima K.; Iwane A.; Yanagida T.

  Biophysics  1998/09  The Biophysical Society of Japan General Incorporated Association
  
• Laser trapping nanometry for single kinesin molecules

  INOUE Y.; HIGUCHI H.; NISHIYAMA M.; IWANE A. H.; IWATANI S.; MIYAI T.; YANAGIDA T.

  Biophysics  1997/10  The Biophysical Society of Japan General Incorporated Association
  
• Detection of nm-displacement of single motor-molecules at hightemporal resolution

  NISHIYAMA M.; H. Hideo; YANAGIDA T.

  Biophysics  1997/10  The Biophysical Society of Japan General Incorporated Association
  

MISC

• Cardiomyocyte contraction measured by high-pressure microscopy

  西山雅祥; 山口陽平; 金子智之; 森松賢順  月刊細胞  55-  (14)  2023
• 高圧力下で蘇る鞭毛運動

  八木俊樹; 西山雅祥  化学  76-  (4)  41  -45  2021/04
• 極限環境を利用した化学の新展開 高圧力技術を用いた生命活動操作イメージング

  西山雅祥  現代化学  (538)  2016
• <研究の現場から>フォースでいのちはあやつれるか？

  西山 雅祥  京都大学白眉センターだより  10-  13  -13  2015/10
• 高度物理刺激と生体応答(5)-第3章 力学刺激による細胞応答と応用 その3-

  西山雅祥  機械の研究  67-  (12)  1069  -1072  2015  [Refereed][Invited]
  
• タンパク質分子機械の力学変調計測

  西山 雅祥  機械の研究  67-  (12)  2015  [Invited]
  
• 細胞内の水で生命活動を操る! : 高圧力下で観るタンパク質水和変調イメージング

  西山 雅祥; 曽和 義幸  化学 = Chemistry  68-  (9)  33  -38  2013/09  [Invited]
  
• 染色体を牽引する「分子エンジン」

  西山 雅祥  67-  (1)  66  -67  2011/12  [Invited]
  
• Realtime measurement of mechanical motion of single protein molecules

  Kenji Okamoto; Masayoshi Nishiyama; Masahide Terazima  2007 INTERNATIONAL SYMPOSIUM ON MICRO-NANO MECHATRONICS AND HUMAN SCIENCE, VOLS 1 AND 2  151  -156  2007  [Refereed]
  
• イオンの流れで駆動する生物分子モーター

  西山 雅祥  月刊化学  62-  (2)  59  -60  2007/01  [Invited]
  
• Development of a high-pressure microscope and its application to biological systems

  Masayoshi Nishiyama; Yoshifumi Kimura; Yoshio Nishiyama; Masahide Terazima  2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS  2006  [Refereed]
  
• Single molecule techniques in biophysics

  T Yanagida; H Tanaka; K Kitamura; T Wazawa; M Nishiyama; S Esaki; Y Sako; T Ide; AH Iwane; Y Ishii  NA/K-ATPASE AND RELATED ATPASES  1207-  71  -85  2000  [Refereed][Invited]
  
• 第38回生物物理若手の会 夏の学校報告

  西山 雅祥  生物物理  38-  (6)  274  -277  1998/11

Industrial Property Rights

• 特願2023-200533:精液の処理方法  2023年/11/28

  松浦 宏治, 西山 雅祥  学校法人 加計学園 岡山理科大学, 学校法人 近畿大学
• 特許5207300:HIGH PRESSURE SAMPLE CONTAINER FOR OPTICAL MICROSCOPIC OBSERVATION    2013/03/01

  西山 雅祥

Awards & Honors

• 2011/04 Minister of Education, Culture, Sports, Science and Technology The Young Scientists’ Prize
   

  受賞者: NISHIYAMA Masayoshi
• 2009/10 Biophysical Society of Japan Early Career Award in Biophysics
   

  受賞者: Masayoshi Nishiyama
• 2001/07 日本工業新聞社主催 先端技術学生論文表彰制度 文部科学大臣賞（最優秀賞）
   JPN 

  受賞者: 西山雅祥
• 2000/09 応用物理学会 Young Scientist Award for the Presentation of an Excellent Paper, The Japan Society of Applied Physics
   JPN

Research Grants & Projects

• Exploration and application of outer membrane vesicle-producing bacteria for the development of novel nanomaterials

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/09 -2027/03 

  Author : 【代表】川本 純; 【分担】西山 雅祥; 豊竹 洋佑
• Development of near-infrared light-responsive flexible nitric oxide-emitting nanomaterials.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2026/03 

  Author : 【代表】小阪田 泰子; 【分担】西山 雅祥; 中川 秀彦
• Visualizing the activation of flagellar beating at high-pressure.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 【代表】西山雅祥; 【分担】八木俊樹; 佐藤裕公; 今井洋; 松浦宏治
• Survival Strategy for Extremophiles around hydrothermal vents

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Transformative Research Areas (A)

  Date (from‐to) : 2022/06 -2024/03 

  Author : 西山 雅祥
• 麻酔薬圧拮抗作用の実時間イメージング

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  Date (from‐to) : 2022/06 -2024/03 

  Author : 【代表】西山雅祥; 【分担】早坂晴子
• 新規メカニカル負荷装置の開発を通した次世代メカノメディスンへの挑戦

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 【代表】成瀬恵治; 【分担】西山雅祥; 高橋賢; 片野坂友紀; 森松 賢順; 入部 玄太郎

   

  1. ストレッチ・静水圧負荷装置の開発：バルク型および顕微鏡型の開発を行った。基本設計は終了しており、微調整を行っている。 2. 軟骨に対するマルチメカニカルストレスへの応答解析：高圧受容応答メカニズムの解明を最終目的とし、高静水圧刺激による細胞及び、細胞内分子の挙動計測を実施した。定常圧力20 MPa以上の圧力を1時間負荷した際、シグナル伝達物質に関わるSmad 3タンパク質の細胞質から細胞核内への核移行が観察された。この圧力に依存したSmad 3の核内移行過程には、TGF-β receptor の活性化やImportin bとの結合が必要であることが分かり、高静水圧に依存したSmad 3核内移行メカニズムの提案が可能となった。 3. 心筋細胞に対するマルチメカニカルストレスへの応答解析：循環器における機械感受性イオンチャネルTRPV2の役割を、組織特異的TRPV2ノックアウトマウスを用いて、明らかにしてきた。その過程で、TRPV2は、心臓への圧負荷依存的肥大や心不全、血管の筋原性緊張や肥厚などに大きく関与する因子であることが明らかとなった。 4. 剪断応力・ストレッチチャンバーでのiPS心筋細胞3次元培養：剪断応力とストレッチの同時刺激が可能な臓器チップを用い、血管内皮細胞を播種した状態で、血管収縮の調節因子である一酸化窒素(NO)のライブイメージングを行った。その結果、ストレッチ刺激および圧力刺激に応じたNOの放出が確認された。 5. 心筋細胞標本の機能評価：心筋細胞のメカニカルストレスとそれに対する応答及び応答伝播の相互関係を観察するため、細胞を直列に配列させる培養法の開発を行った。フォトエッチング技術により描画した直線状パターンを鋳型としたPDMS製のマイクロ流路を作製し、これをイオンボンバーダーで親水処理した。これを用いてマウス幼若心筋細胞を播種することで、細胞を一次元的に配向させた状態で培養することに成功した。
• Mechanical control of rhythmic flagellar beating motions

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : Nishiyama Masayoshi

   

  The beating of eukaryotic flagella depends on the sliding movements between microtubules powered by dynein. In flagella of most organisms, microtubule sliding is regulated by the internal structure of flagella comprising the central pair of microtubules (CP) and radial spokes (RS). Chlamydomonas paralyzed-flagella (pf) mutants lacking CP or RS are non-motile under physiological conditions. Here, we show that high hydrostatic pressure induces vigorous flagellar beating in pf mutants. The beating pattern at 40 MPa was similar to that of wild type at atmospheric pressure. In addition, at 80 MPa, flagella underwent an asymmetric-to-symmetric waveform conversion, similar to the one triggered by an increase in intra-flagella Ca2+ concentration during cell’s response to strong light. Thus, our study establishes that neither beating nor waveform conversion of flagella requires the presence of CP/RS in the axoneme.
• Manipulating nano-structures with hydrostatic pressures and optical forces

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2019/04 -2021/03 

  Author : 西山 雅祥

   

  本研究の目的は、ナノメートルスケールの構造体を操作する新しいマニピュレーション技術の創出にある。研究代表者は、光の放射圧を用いて微小物体の操作を行える「光圧」技術と、等方的で一様な力学作用である「圧力」を変える技術に着目した。これまでは、全く異なる手法として認知されてきたこれらの技術を融合させることが骨子である。令和元年度には、１気圧から0.15気圧まで減圧できるチャンバーを開発し、その中を観察できる減圧顕微鏡を開発した。令和二年度は、この減圧顕微鏡に、高開口数の油浸対物レンズと高出力かつ直進性の高い近赤外レーザー光源を組み合わせて、光ピンセットの光学系を再構築した。光学系が改良されたことで、光源の出力に対して、光捕捉の力をより向上させることができた。この装置を用いて、大腸菌を用いた性能チェックを行った。大腸菌は細胞の周りに生やしたべん毛を船のスクリューのように回転させて推進力を発生させている。開発した装置を用いて、水の中を泳ぐ大腸菌を光ピンセットで捕捉し、その後、開放することができた。捕捉前後で遊泳速度に変化がないことから、非侵襲での捕捉できたことになる。また、減圧し0.15 気圧にしたところ、水の中を泳ぐ大腸菌の割合と速度が大幅に低下した。減圧下にある水溶液中の酸素濃度はヘンリーの法則に従い減少することから、大腸菌は酸欠により運動能を低下させたと考えられる。高圧力下での光捕捉については、耐圧性能と光学性能を両立させた高圧力チャンバーを開発する必要があり、現在、継続中である。
• High-resolution imaging of intermolecular interactions undergoing mechanical modulation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2020/03 

  Author : Nishiyama Masayoshi

   

  Application of hydrostatic pressure is a powerful method for modulating intermolecular interactions between protein and water molecules. We have developed a high-pressure microscope that is optimized both for the best image formation and for the stability to hydrostatic pressure up to 150 MPa. The developed apparatus allows us to visualize the structural and functional changes of molecular machines. By using high-pressure microscopy, we have developed a novel technique that controls the molecular machines working in living cells. The developed technique successfully demonstrated that application of pressures can increase the activity in living cells.
• Reconsitutive studies on molecular mechanisms underlying regulation of rotational molecular machines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2017/07 -2020/03 

  Author : Kawagishi Ikuro

   

  The bacterial flagellar motor is a nanometer-size rotary molecular machine driven by ion flux. One remarkable feature of the motor is that its rotation can be instantaneously switched from counterclockwise to clockwise upon binding of the active form of the cytoplasmic signaling protein CheY. Here we found that higher hydrostatic pressure weakens the CheY-FliM interaction, demonstrating that is a critical step for the regulation of motor rotation. We also showed that in bacteria with a polar flagellum, cooperativity of the CheY-FliM interaction is tuned to be low and that this is a key feature essential for repellent responses. Our finding sheds new light on the motor switching mechanism.
• 大腸菌べん毛モーターの回転コントロールによるスイッチング機構の解明

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2017/04 -2019/03 

  Author : 西山 雅祥

   

  本研究課題の目的は、細菌べん毛モーターの回転方向がどのように制御されているのかその分子機構を明らかにすることにある。研究代表者はこれまで開発してきた高圧力顕微鏡法と細菌運動観察の手法を用いて、平成３０年度は下記の研究を実施した。 １）細菌は、細胞外の環境変化を知覚し、細胞内にある走化性応答制御因子CheYをリン酸化することで、モーターへの結合能を上昇させる。この活性化型CheYがモーターに一定数結合するとモーターの回転方向が反転することになる。細胞外環境にかかわらず、常時、活性化型となるCheY変異体を作成し実験を行った。常圧力下では十分量のCheYがモーターに結合しCW方向に安定して結合していた。それに対して、圧力値の増加すると、CW方向への回転方向性を示すCWbiasが急峻に低下しほぼゼロとなった。これは、高圧力によりモーターと活性化型CheYとの結合量が調節出来たことを強く示唆される。次に、CW biasと回転速度との相関を調べたところ、CWbiasが0.5近くでは、回転速度が約半分に低下することが明らかになった。これは、CheYの結合はモーターの回転方向のみならず、トルク発生にも大きく関与していることを示す結果である。 ２）磁性細菌MO-1は２つのべん毛運動マシナリ－を使って、地磁気に沿って泳ぐ性質がある。その際に見られる運動の軌跡はらせん状となっている。この特異な運動様式がもつ意味を調べるために、平面近くでの磁性細菌の泳ぎを調べたところ、この運動様式には平面から遠ざかる性質があることが判明した。つまり、MO-1はこの特異な運動様式をとることで、生育環境に多数存在する砂粒表面に沿った動きを避けながら、地磁気に沿った泳ぎを達成させていることが明らかになった。
• 極限環境下にある深海微生物の生存戦略イメージング

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2015/04 -2017/03 

  Author : 西山 雅祥

   

  研究代表者はこれまでから高圧力環境にある生物試料を実時間で観察できる高圧力顕微鏡の開発を行ってきた。この装置を利用すると、地球上で最も深い領域であるマリアナ海溝最深部（約11,000m）の静水圧を超える圧力環境を研究室で作り出すことができる。平成27年度は、海洋研究開発機構（JAMSTEC）が有人潜水調査船「しんかい６５００(最大潜航深度6,500m) により深海底から採取してきた海洋細菌の運動マシナリーを調べる研究を行った。Shewanella violacea DSS12は琉球海溝深度5,000mから採取された代表的な海洋細菌である。8 ℃, 30 MPaを生育至適とし、70MPa程度の高圧環境にまで良好に生育できることから好圧菌のモデル生物とされている。また、これまでの研究によりゲノム解析も達成されている（4.8 Mb）。低温（８℃）、大気圧環境下でこの菌体を対数増殖期まで培養し、高圧力環境下で遊泳速度の圧力依存性を調べた。８℃, 0.1MPa条件下では、約半分の菌体が遊泳運動を行っており、その速度は約16 μm/sであった。圧力の増加に伴い、泳ぐ菌体の割合と速度は単調に減少し、100 MPaで共に約半分になった。大気圧環境下で生育する大腸菌は23℃, 80MPaでは完全に遊泳運動を停止したことから、圧力変化に対するDSS12株の遊泳運動能の変化は緩やかであり、高圧力環境への適応が示唆された。
• High-pressure microscopy for controlling the structure of protein filaments

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2013/04 -2016/03 

  Author : Nishiyama Masayoshi

   

  We have developed a high-pressure microscope that enables to observe the structure and function of protein molecules. In this research project, we have studied the conformational changes of protein filaments at high-pressure. By using this system, we were successful to change the helical structure of flagellar filaments and microtubule dynamics.
• 極限環境下にある超好熱始原菌の運動観察

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2013/06 -2015/03 

  Author : 西山 雅祥

   

  超好熱菌は至適生育温度が80°C以上の微生物として定義されており、古細菌の多くに加えて、真正細菌の中ではAquifex属などが含まれている。Aquifex属は16S rRNA系統解析から真正細菌の中でも最も初期に分岐したことが示されている。研究代表者がこれまで開発してきた装置を用いれば、高温、高圧、嫌気条件といった極限環境を顕微鏡下につくりだし、菌体の様子をリアルタイムで観察できる。平成26年度は、真正細菌の中で遺伝的・構造的に広く保存された運動器官であるべん毛に着目し、Aquifex属の代表例であるA. aeolicusの運動マシナリーを調べる研究を行った。Huber博士から提供していただいたA. aeolicusの野生株を対数増殖期まで生長させ、培地内での遊泳運動を顕微観察した。その結果、多くのA. aeolicusは遊泳運動を示さなかったものの、一部の菌体は培地内をほぼまっすぐに泳ぐことが明らかになった。その遊泳速度は至適生育温度である85℃では約90µm/sであり、その速度は温度とともに低下した。菌体の電子顕微鏡撮影を行ったところ、少数の菌体からは細胞の片方の極から主として１本の鞭毛繊維が生えているのが観察された。多くの細胞本体からはべん毛繊維は見られなかったものの、この結果は光学顕微鏡下で遊泳運動を示す菌体が少なかったことと一致する。以上の結果から、A. aeolicusは細胞の局部にあるべん毛繊維を運動マシナリーとして利用して遊泳運動を行っていると考えられる。
• 好熱菌由来ＡＴＰ駆動型分子モーターの運動測定

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2011/04 -2013/03 

  Author : 西山 雅祥

   

  ATP合成酵素F1-ATPaseは、ATPを加水分解しながら回転運動を行う代表的な分子モーターである。本研究課題では、研究代表者がこれまでより開発した高圧力顕微鏡を用いて(Nishiyama and Sowa, Biophys J. (2012))、高圧力にあるF1-ATPaseの回転運動がどのように変化するのか調べてきた。平成２４年度は、回転運動の変位トレースの詳細な解析を行った。高圧力下にある野生型のF1-ATPaseの回転トレースは、ATP濃度に依存せず120#186; を単位とするステップ状の変位から構成されており、80#186;と40#186;のサブステップは検出されなかった。また、120#186;ステップの立ち上がり速度には圧力依存性がみられなかったので、高圧力下であってもF1-ATPaseの構造やトルク発生にいたるポテンシャル形状にはほぼ影響がなかったことを意味する。その後の解析により、40#186;サブステップから80#186;サブステップまでのATP binding dwellに圧力依存性があることが判明し、ATPの結合反応、および、それに続く（１つ以上の）反応過程が高圧力の影響をうけることが明らかになった。これらの活性化体積は、それぞれ+100#197;3と+88#197;3であり、水分子３個分程度の体積変化に相当すする。今後、圧力の影響を顕著に受けている中間状態の構造が解明されれば、高圧力条件下で水を含んだＭＤ計算を実施することで、F1-ATPaseの水和状態がどのようにして回転運動を生み出しているのか、その詳細なメカニズムが明らかになると期待される。
• Visualization of Mechanical Response of Molecular Machines working in vivo

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2011 -2012 

  Author : NISHIYAMA Masayoshi

   

  In this research project, we developed a new assay that enables to control the functional expression of molecular machines working in living cells. We constructed a new high-pressure microscope that allows us to modulate the intermolecular interactions between protein and water molecules. By using this system, we were successful to change the rotational direction of Escherichia coli flagellar motors.
• ATP駆動型分子モーターの圧力変調と運動解析

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2009 -2011 

  Author : 西山 雅祥

   

  細胞内にあるタンパク質などの生体分子は、周囲を水分子に取り囲まれ水素結合を形成し、エネルギー的な最適化がほどこされている。本研究課題では、高圧力技術を用いてタンパク質の水和状態を変えることで、ATP駆動型分子モーターの運動教変調を行うことが目的である。H22年度では下記の研究を行った。 A)市販の光学顕微鏡にそのまま登載できる高圧力顕微鏡の開発。従来までの高圧力顕微鏡では、手押しポンプで発生した水圧を、高圧容器に封入したバッファー圧に変換するための外部セパレーターが必要であり、10ml近いバッファーを準備する必要があった。また、高圧チャンバーを固定するための専用の顕微鏡ステージが必要としていた。本研究課題では、高圧チャンバー内でポンプの水圧をバッファーの圧力に変換するセパレーター機能を内在した顕微観察用のミニチュアチャンバーを作成した。この内部セパレーター機能を採用することで、高圧装置の全体のサイズを大幅に下げることに成功し、市販の光学顕微鏡本体に付随するスライドグラスの固定器具を利用して、簡便に顕微観察ができる仕様にした。この装置を用いて、実際に高圧力下での大腸菌の遊泳運動観察を行い、従来までのシステムと同等の性能を有することを確認した。 B)市販の吸光光度計、蛍光分光器に搭載できる高圧チャンバーの開発。市販の分光器に搭載し、温度と圧力を変えながら、吸収もしくは、蛍光スペクトルを取得できる簡便な装置を開発した。これにより、高圧力下において、蛍光性タンパク質や大腸菌内に取り込ませたNa指示薬の分光測定を実施した。
• ロータリー型分子モーターのトルク発生コントロールと運動計測

  日本学術振興会：科学研究費助成事業 特定領域研究

  Date (from‐to) : 2009 -2010 

  Author : 【代表】西山雅祥; 【分担】曽和義幸

   

  バクテリア・べん毛モーターは、細胞内に流入するイオン流により、回転運動を生み出すタンパク質分子機械である。菌体は、この回転器官を用いて、細胞外に長く伸びたべん毛繊維をスクリューのように回転させることで推進力を発生し、遊泳運動を行っている。本研究課題では、タンパク質の構造と機能を変調できる高圧力技術を利用して、べん毛モーターの高速回転運動の変調と検出により、回転機構を明らかにする研究に取り組んだ。平成21年度の研究により、高圧力下では、細胞内へのH+の流入速度が低下することが判明した。そこで、平成22年度では、Na+の流入により駆動するキメラべん毛モーターを用いて、細胞外のNa+濃度を変化させながら、回転計測を行った。べん毛繊維にポリスチレンビーズを回転の指標として吸着させ、ハイスピードカメラを搭載した高圧力顕微鏡でビーズの高速回転運動を観察した。常圧力下では、モーターの回転速度は、細胞外のNa+濃度と共に増加し、85mMNa+では、600Hzに達した(0.4umビーズ)。高圧力下では、全てのNa+濃度下で、圧力の増加共に回転速度は指数関数的に減少した。以上の結果は、H+駆動型べん毛モーターから得られた実験結果と一致するため、高圧力はNa+の流入速度を低下させていることが明らかになった。次に、蛍光指示薬を用いて、高圧力下にある細胞内Na+濃度を調べたところ、大きな変化はなかった。したがって、高圧力をかけることで、細胞外にあるNa+がモーターへの結合反応が阻害されることで、回転速度が低下すると考えられる。
• Manipulation and nano-measurement of the structural fluctuation of biomolecules.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2008 -2009 

  Author : MASAYOSHI Nishiyama

   

  Protein hydration is an important factor for structural formation and enzyme activity. To study the effects of hydration, it is desirable to monitor protein structures or biological activities by modulating the intermolecular interaction between protein and water molecules. In this study, we have developed a novel microscopy for visualizing the pressure-induced changes in the structure and function of biomolecules. We demonstrated that the rotational movement of molecular motors is successively detected with nanometer spatial resolution at high pressure.
• Molecular mechanisms of various side effects of volatile anesthetics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2007 -2009 

  Author : MIYAMOTO Yoshikazu; IWANE Atsuko; YANAGIDA Toshio; MAHIMO Takashi; NISHIYAMA Masayoshi

   

  By using in vitro motility assay, we determined the direct effects of inhalational anesthetics sevoflurane and isoflurane on microtubule-based kinesin motility. By applying inhalational anesthetics, most of the microtubules stopped sliding movements, and their motility almost recovered after washing out of the anesthetics.
• 高圧力顕微鏡の開発と生体システムへの応用

  日本学術振興会：科学研究費助成事業 特定領域研究

  Date (from‐to) : 2006 -2007 

  Author : 【代表】西山雅祥; 【分担】岡本憲二

   

  細胞内に含まれる成分のうち、70%は水であり、生命活動を担う多くの化学反応は水溶液中で進行している。本研究課題では、タンパク質、DNAをはじめとする生体分子が、周囲をとりまく水分子と相互作用することで、どのようにして生物らしい構造形成や機能発現を生み出しているのかそのメカニズムを明らかにすることを目的としている。研究代表者らは、タンパク質の水和状態に着目し、高圧力により水和状態を変化させることで、構造・機能の変調イメージングを行える高圧力顕微鏡の開発に取り組んできた。H19年度は、バクテリアの運動器官であるべん毛モーターを研究対象に、高圧力による回転運動変調と応答計測を行った。べん毛モーターは、主要部分だけでも200以上のタンパク質から構成される巨大なタンパク質複合体であり、細胞外から流入するプロトンイオンの流れを利用して、トルク発生を行っている。研究代表者らは、べん毛の回転方向を調節するCheYを欠失させた菌体を用いて、高圧力顕微鏡の観測窓にべん毛を吸着させ「テザードセル」の実験系を構築し、回転運動が圧力によりどのように変調されるのか観測した。その結果、1000気圧程度の圧力により、べん毛モーターの回転方向・速度が変調されることが明らかになった。このような高圧力下でみられる運動様式は、べん毛モーターのトルク発生部位の周辺の構造や相互作用様式が変化することで引き起こされていると考えられる。
• 1分子イメージングのための高圧力蛍光顕微鏡の開発

  日本学術振興会：科学研究費助成事業 萌芽研究

  Date (from‐to) : 2006 -2007 

  Author : 【代表】木村佳文; 【分担】西山 雅祥

   

  本研究の目的は、タンパク質に圧力をかけることで引き起こされる構造変化や機能変調の1分子計測を可能にする顕微観測システムを開発することにある。これまでの研究により、タンパク質間の分子間力は、数千気圧程度の圧力により変調をきたすことが知られている。平成19年度は、タンパク質間相互作用の変調を可能とする2000気圧程度の耐圧性能を保持しながら、高解像度の顕微観測を可能とする高圧セルの開発に取り組んだ。汎用性の高い実験系とするために、市販の光学顕微鏡を用いて実験できることが条件となる。そこで、高圧セルの本体には、1)高強度材料であるハステロイ鋼を利用した単純な構造とし、2)部品数を減らしコンパクトな構造であり(70×70×23mm)、O-ringで簡便に圧力シールができるよう仕様にした。また、恒温槽を用いた循環水で簡便に温度を変化させられるようにした。高解像度の顕微観測を行うためには、開口数の高い油浸対物レンズを利用することが必要不可欠であり、高圧セルの観測窓の薄型化(〜0.2mm)に迫られる。そこで、研究代表者らは観測窓を小さくすることで(〜0.2mm)、耐圧性能と高開口数の両立をはかることにした。観測窓周辺のデザインや加工精度により、耐圧性能にばらつきは生じるものの、おおむね2000気圧程度の耐圧性能を達成させることに成功した。しかしながら、現状のデザインでは、高度な試料調整を伴う生体分子の1分子イメージングを実行することは困難であり、今後観測窓表面を適切に処理できるように改良を加え実用化に向けて検討を重ねる予定である。
• 生物分子モーター1分子のメガヘルツ応答計測と熱ラチェット型メカニズムの解明

  日本学術振興会：科学研究費助成事業 若手研究(B)

  Date (from‐to) : 2006 -2007 

  Author : 西山 雅祥

   

  本研究の目的は、生物分子モーターがどのようにして、化学エネルギーを力学エネルギーへと変換しているのか、その仕組みを明らかにすることにある。キネシンは、細胞内の物質輸送を担う代表的な分子モーターであり、ATPを加水分解しながら微小管に沿ってステップ状の変位を発生させている。研究代表者はこれまでより、光ピンセットを用いたナノメートル計測装置を用いて、キネシン1分子の力学計測を行ってきた。本年度は、1)従来よりも分解能を向上させたナノメートル計測系の構築と、2)キネシンのステップ状変位を元にした運動解析を行った。前者については、従来までの分解能では捉えられなかった変位検出を目標に、分子モーターの動きの指標となるビーズからS/Nの高い像を形成させる暗視野照明型の光学系開発に取り組んだ。レーザー光をリング状に拡げた輪帯照明の光学系を構築したが、現状ではビーズから得られる散乱強度が不十分であり、さらなる光学系の改良が必要である。また、後者については、様々なATP濃度で計測したキネシンのステップ状変位の解析を行ったところ、キネシンが前後にステップする比にはATP濃度依存性が見られ、全ての力領域において、低ATP濃度下では前方方向にステップする頻度が少なくなる傾向が見られた。これは、力による微小管からの解離現象が含まれることに起因する。モデル解析を行ったところ、キネシンは負荷に抗して運動している際には一つのモータードメインを介して微小管に結合していることが示唆された。
• Study on adaptation of single photosynthetic cells against environmental stress by using three-dimensional super-resolution microscopy with time resolution ability

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas

  Date (from‐to) : 2004 -2006 

  Author : KUMAZAKI Shigeichi; NISHIYAMA Masayoshi; OH-OKA Hirozo

   

  A near-infrared fs pulse was illuminated on a line in a specimen by a resonant scanning mirror oscillating at 7.9kHz, and total multiphoton-induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron-multiplying CCD camera was used to resolve fluorescence of different colors at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. The full widths at half maximum of the point-spread function of the system were estimated to be 0.39-0.40, 0.33 and 0.56-0.59μm for the X, Y and Z axes, respectively, at fluorescence wavelengths between 644 and 690nm. This microscope was applied to a study of the subcellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of subcellular domains. Adaptation of the thylakoid membrane against light conditions, and its variation between chloroplasts and cyanobacteria, has been known for decades, but characterization based on fluorescence spectrum at a subcellular resolution has been scarce. Our newly developed spectroimaging system has also been applied to chloroplasts in tabacco, zea mays, and chlorella. Tissue dependence in the case of tobacco, daily cycle in the case of zea mays, and culture-condition dependence in the case of chlorella were found.
• 生体分子機械の構造揺らぎと機能発現のメカニズムの解明

  日本学術振興会：科学研究費助成事業 若手研究(B)

  Date (from‐to) : 2003 -2004 

  Author : 西山 雅祥

   

  タンパク質の天然構造は、数多くの準安定構造との平衡状態にあり、非常に揺らぎやすい性質をもっている。この「柔らかな」立体構造には、タンパク質が機能発現に際して引き起こす大きな構造変化と密接な関わりがあると考えられており、これまでから、高圧NMRなどを用いて、タンパク質構造が大きく揺らぐ部位の同定がなされてきている。研究代表者は、高圧下で生体高分子フィラメントの構造揺らぎを制御しながら、顕微観測を可能にする「高圧力蛍光顕微鏡」を開発した。小型の高圧光学容器(ステンレス製:5.2x5.2x3.4cm)には2つの観測窓が空けてあり、容器内に封入した0.17mlのサンプル溶液を観測できる。観測窓の窓材には、耐圧性と高解像度の顕微観測を両立させるため、厚さ0.5mmのダイヤモンドを採用し光軸方向の厚みをおさえた。この高圧容器を倒立型顕微鏡(IX71, Olympus)にとりつけ、長作動距離の対物レンズ(SLCPlanFl 40x, Olympus)を用いて顕微観測したところ、蛍光染色した微小管の落射蛍光像を観察することに成功した。100MPa(1000気圧)の圧力下で、キネシン-微小管系のin Vitro Motility Assayを行ったところ、滑り運動が20%低下することが明らかになった。今後は、多くのタンパク質が変性しはじめる400MPaの耐圧性をもつ高圧セルを開発すると共に、圧力添加による構造揺らぎと機能発現過程との相関を明らかにする研究を行っていく。
• 生体分子ダイナミクスに関する研究

  科学研究費補助金

  Date (from‐to) : 2002
• Study on Dynamic Properties of Biomolecules

  Grant-in-Aid for Scientific Research

  Date (from‐to) : 2002
• 単一生体分子内のオングストロームの動きを見る顕微鏡の開発

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  Date (from‐to) : 1998 -2000 

  Author : 西山 雅祥

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https://researchmap.jp/read0093328