Researcher Database

Hisashi Haga
Faculty of Advanced Life Science Advanced Transdisciplinary Science Cellular Dynamics Science
Professor

Researcher Profile and Settings

Affiliation

  • Faculty of Advanced Life Science Advanced Transdisciplinary Science Cellular Dynamics Science

Job Title

  • Professor

Degree

  • Ph.D., Science(Hokkaido University)

Research funding number

  • 00292045

J-Global ID

Research Interests

  • 力学的メモリー効果   表面粘性   表面剛性   細胞形態   TGFB-1   リン酸化   radiation   irradiation   放射線感受性   細胞生物学   生物物理学   Biological Physics   Cell Biology   カンチレバー   放射線   Radiotherapy   粘弾性   走査型プローブ顕微鏡   上皮細胞   コラーゲンゲル   細胞運動   集団運動   細胞培養   結合組織   組織形成   接着構造   ゲル基質   メカノセンス   細胞   放射線治療   

Research Areas

  • Natural sciences / Bio-, chemical, and soft-matter physics
  • Life sciences / Morphology, anatomy
  • Life sciences / Cell biology
  • Life sciences / Biophysics
  • Life sciences / Tumor biology

Academic & Professional Experience

  • 2013/05 - Today Hokkaido University Faculty of Advanced Life Science
  • 2010 - 2013 - 北海道大学 大学院先端生命科学研究院 准教授 准教授
  • 2007 - 2010 Hokkaido University Graduate School of Science, Division of Biological Sciences
  • 2006 - 2007 Hokkaido University Graduate School of Science, Division of Biological Sciences
  • 2006 - 2007 Associate Professor
  • 2002 - 2006 北海道大学 大学院理学研究科生物科学専攻 助教授 助教授
  • 2002 - 2006 Associate Professor
  • 1997 - 2002 北海道大学 大学院理学研究科物理学専攻 助手 助手
  • 1997 - 2002 Research Associate
  • 1995 - 1997 米国マサチューセッツ工科大学 化学科 博士研究員 研究員
  • 1995 - 1997 Researcher
  • 1994 - 1995 日本学術振興会 特別研究員 日本学術振興会特別研究員
  • 1994 - 1995 Postdoctoral Fellowships of Japan Society for the Promotion of Science
  • 1992 - 1995 北海道大学 教養部 非常勤講師 講師
  • 1992 - 1995 Lecturer

Education

  • 1992/04 - 1995/03  Hokkaido University
  •        - 1995  Hokkaido University  Graduate School, Division of Natural Science
  • 1990/04 - 1992/03  Hokkaido University
  •        - 1992  Hokkaido University  Graduate School, Division of Natural Science
  • 1986/10 - 1989/03  Hokkaido University  School of Science  Physics
  •        - 1989  Hokkaido University  Faculty of Science
  • 1985/04 - 1986/09  Hokkaido University

Association Memberships

  • The Japanese Society of Mechanobiology   日本細胞生物学会   日本生物物理学会   米国細胞生物学会(ASCB)   日本癌学会   Japanese Society for Cell Biology   American Society for Cell Biology   

Research Activities

Published Papers

  • Sumire Ishida-Ishihara, Masakazu Akiyama, Kazuya Furusawa, Isao Naguro, Hiroki Ryuno, Takamichi Sushida, Seiichiro Ishihara, Hisashi Haga
    Journal of cell science 2020/06/23 [Refereed][Not invited]
     
    One of the fundamental processes of morphogenesis is dome formation, but many parts of the mechanisms has been unexplored. Previous in vitro studies showed that osmotic gradient is the driving factor of the dome formation. However, these investigations were performed without extracellular matrix (ECM), which provides structural support to morphogenesis. With the use of ECM, we observed that basal hypertonic stress induced stable domes in vitro that have not been seen in previous studies. These domes developed from the ECM swelling via aquaporin water transport activity. Based on computer simulation, uneven swelling, with a positive feedback between extending cell and enhanced water transport, was a cause for dome formation. These results indicate that osmotic gradient induces dome morphogenesis via both enhanced water transport activity and subsequent ECM swelling.
  • Mizutani Y, Kobayashi H, Iida T, Asai N, Masamune A, Hara A, Esaki N, Ushida K, Mii S, Shiraki Y, Ando K, Weng L, Ishihara S, Ponik SM, Conklin MW, Haga H, Nagasaka A, Miyata T, Matsuyama M, Kobayashi T, Fujii T, Yamada S, Yamaguchi J, Wang T, Woods SL, Worthley D, Shimamura T, Fujishiro M, Hirooka Y, Enomoto A, Takahashi M
    Cancer research 79 (20) 5367 - 5381 0008-5472 2019/10 [Refereed][Not invited]
  • Frauenlob Martin, King Daniel R, Guo Honglei, Ishihara Seiichiro, Tsuda Masumi, Kurokawa Takayuki, Haga Hisashi, Tanaka Shinya, Gong Jian Ping
    MACROMOLECULES 52 (17) 6704 - 6713 0024-9297 2019/09/10 [Refereed][Not invited]
  • Kumagai Y, Nio-Kobayashi J, Ishida-Ishihara S, Tachibana H, Omori R, Enomoto A, Ishihara S, Haga H
    Biochemical and biophysical research communications 514 (4) 1115 - 1121 0006-291X 2019/07 [Refereed][Not invited]
  • Yamamoto K, Otomo K, Nemoto T, Ishihara S, Haga H, Nagasaki A, Murakami Y, Takahashi M
    Experimental cell research 376 (1) 67 - 76 0014-4827 2019/03 [Refereed][Not invited]
  • Acebedo AR, Suzuki K, Hino S, Alcantara MC, Sato Y, Haga H, Matsumoto KI, Nakao M, Shimamura K, Takeo T, Nakagata N, Miyagawa S, Nishinakamura R, Adelstein RS, Yamada G
    Communications biology 2 95  2019 [Refereed][Not invited]
  • Wang X, Enomoto A, Weng L, Mizutani Y, Abudureyimu S, Esaki N, Tsuyuki Y, Chen C, Mii S, Asai N, Haga H, Ishida S, Yokota K, Akiyama M, Takahashi M
    Cancer science 109 (11) 3643 - 3656 1347-9032 2018/11 [Refereed][Not invited]
  • Koh I, Furusawa K, Haga H
    Scientific reports 8 (1) 13901  2018/09 [Refereed][Not invited]
  • H. Oyama, K. Takahashi, Y. Tanaka, H. Takemoto, H. Haga
    Cell Structure and Function 43 (1) 85 - 94 2018/05 [Refereed][Not invited]
  • Ishihara S, Aoki K, Mizutani T, Amano M, Nishimura SI, Haga H
    Cell structure and function 43 (2) 177 - 185 0386-7196 2018 [Refereed][Not invited]
  • Ishihara S, Ponik SM, Haga H
    Oncoscience 4 (11-12) 158 - 159 2017/11 [Refereed][Not invited]
  • Akiyama M, Sushida T, Ishida S, Haga H
    Development, Growth & Differentiation 59 (5) 471 - 490 2017/07 [Refereed][Not invited]
  • Ayuko Sakane, Shin Yoshizawa, Masaomi Nishimura, Yuko Tsuchiya, Natsuki Matsushita, Kazuhisa Miyake, Kazuki Horikawa, Issei Imoto, Chiharu Mizuguchi, Hiroyuki Saito, Takato Ueno, Sachi Matsushita, Hisashi Haga, Shinji Deguchi, Kenji Mizuguchi, Hideo Yokota, Takuya Sasaki
    MOLECULAR BIOLOGY OF THE CELL 27 (20) 3095 - 3108 1059-1524 2016/10 [Refereed][Not invited]
     
    In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides "law and order" in collective cell migration.
  • Akihiro Nukuda, Hiroki Endoh, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 474 (3) 509 - 514 0006-291X 2016/06 [Refereed][Not invited]
     
    Activating transcription factor 5 (ATF5) is a member of the ATF/CAMP response element-binding protein family. Our research group recently revealed that ATF5 expression increases the invasiveness of human lung carcinoma cells. However, the effects of ATF5 on the invasive potential of other cancer cells lines remain unclear. Therefore, in this study, we investigated the role of ATF5 in the invasive activity of diverse human cancer cell lines. Invasiveness was assessed using Matrigel invasion assays. ATF5 knockdown resulted in decreased invasiveness in seven of eight cancer cell lines tested. These results suggest that ATF5 promotes invasiveness in several cancer cell lines. Furthermore, the roles of ATF5 in the invasiveness were evaluated in three-dimensional (3D) culture conditions. In 3D collagen gel, HT-1080 and MDA-MB-231 cells exhibited high invasiveness, with spindle morphology and high invasion speed. In both cell lines, knockdown of ATF5 resulted in rounded morphology and decreased invasion speed. Next, we showed that ATF5 induced integrin-alpha 2 and integrin-beta 1 expression and that the depletion of integrin-alpha 2 or integrin-beta 1 resulted in round morphology and decreased invasion speed. Our results suggest that ATF5 promotes invasion by inducing the expression of integrin-alpha 2 and integrin-beta 1 in several human cancer cell lines. (C) 2016 Elsevier Inc. All rights reserved.
  • Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    DATA IN BRIEF 6 793 - 798 2352-3409 2016/03 [Refereed][Not invited]
     
    This data article describes cellular dynamics, such as migration speed and mobility of the cytoskeletal protein, of wild-type human fibroblast cells and cells with a modified adeno-associated virus integration site 1 (AAVS1) locus on human chromosome 19. Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1-modification changes cellular contractile force (Mizutani et al., 2015 [1]). To assess if this AAVS1-modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article. Cell nuclei were stained and changes in their position attributable to cell migration were analyzed. Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85 [1]. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CCBY license.
  • Takeomi Mizutani, Kazuya Furusawa, Hisashi Haga, Kazushige Kawabata
    REGENERATIVE THERAPY 3 90 - 96 2352-3204 2016/03 [Refereed][Not invited]
     
    Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are expected to play an important role in heart therapies, in which hiPSC-CMs should generate sufficient contractile force to pump blood. However, recent studies have shown that the contractility of myocardial mimics composed of hiPSC-CMs is lower than that of adult human myocardium. To examine the mechanism by which contractile force output of hiPSC-CMs is weakened, we measured the contractile force of single hiPSC-CMs and observed the fibrous distribution of myosin II regulatory light chain (MRLC) of cardiac (contributes to beating) and non-cardiac (does not contribute to beating) isoforms. Single hiPSC-CMs were cultured on an extracellular matrix gel, and the contractile force and strain energy exerted on the gel were measured. Strain energy was not uniform between cells and ranged from 0.2 to 5.8 pJ. The combination of contractile force measurement and immunofluorescent microscopy for MRLC isoforms showed that cells with higher strain energy expressed the weakened non-cardiac myosin II fibers compared to those of cells with lower strain energy. Observation of cardiac and non-cardiac MRLC showed that the MRLC isoforms formed heterogeneous filament networks. These results suggest that strain energy output from single hiPSC-CMs depends both cardiac and non-cardiac myosin fibers, which prevent deformation of the cell body. (c) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.
  • Tamaki Yamada, Masumi Tsuda, Takanori Wagatsuma, Yoichiro Fujioka, Mari Fujioka, Aya O. Satoh, Kosui Horiuchi, Shinya Nishide, Asuka Nanbo, Yasunori Totsuka, Hisashi Haga, Shinya Tanaka, Masanobu Shindoh, Yusuke Ohba
    SCIENTIFIC REPORTS 6 (56878) 1 - 16 2045-2322 2016/03 [Refereed][Not invited]
     
    Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-kappa B ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin alpha 2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin alpha 2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-kappa B signaling mediated integrin alpha 2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin beta 1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin alpha 2 and endocytosis. Moreover, the RANK-integrin alpha 2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.
  • Seiichiro Ishihara, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga
    CYTOTECHNOLOGY 68 (1) 25 - 32 0920-9069 2016/01 [Refereed][Not invited]
     
    Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-kappa B family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.
  • Takeomi Mizutani, Rui Li, Hisashi Haga, Kazushige Kawabata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 465 (2) 270 - 274 0006-291X 2015/09 [Refereed][Not invited]
     
    The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVSI (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force. (C) 2015 Elsevier Inc. All rights reserved.
  • A. Nukuda, C. Sasaki, S. Ishihara, T. Mizutani, K. Nakamura, T. Ayabe, K. Kawabata, H. Haga
    ONCOGENESIS 4 e165  2157-9024 2015/09 [Refereed][Not invited]
     
    Abnormally stiff substrates have been shown to trigger cancer progression. However, the detailed molecular mechanisms underlying this trigger are not clear. In this study, we cultured T84 human colorectal cancer cells on plastic dishes to create a stiff substrate or on collagen-I gel to create a soft substrate. The stiff substrate enhanced the expression of matrix metalloproteinase-7 (MMP-7), an indicator of poor prognosis. In addition, we used polyacrylamide gels (2, 67 and 126 kPa) so that the MMP-7 expression on the 126-kPa gel was higher compared with that on the 2-kPa gel. Next, we investigated whether yes-associated protein (YAP) affected the MMP-7 expression. YAP knockdown decreased MMP-7 expression. Treatment with inhibitors of epidermal growth factor receptor (EGFR) and myosin regulatory light chain (MRLC) and integrin-alpha 2 or integrin-beta 1 knockdown downregulated MMP-7 expression. Finally, we demonstrated that YAP, EGFR, integrin-alpha 2 beta 1 and MRLC produced a positive feedback loop that enhanced MMP-7 expression. These findings suggest that stiff substrates enhanced colorectal cancer cell viability by upregulating MMP-7 expression through a positive feedback loop.
  • Misako Imai, Kazuya Furusawa, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga
    SCIENTIFIC REPORTS 5 (14208) 1 - 10 2045-2322 2015/09 [Refereed][Not invited]
     
    Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.
  • Seiichiro Ishihara, Hisashi Haga
    AGING-US 7 (7) 453 - 454 1945-4589 2015/07 [Refereed][Not invited]
  • Iguchi Y, Ishihara S, Uchida Y, Tajima K, Mizutani T, Kawabata K, Haga H
    Cell Structure and Function 40 (2) 67 - 67 0386-7196 2015/04 [Refereed][Not invited]
  • Kenji Takemoto, Seiichiro Ishihara, Takeomi Mizutai, Kazushige Kawabata, Hisashi Haga
    PLOS ONE 10 (3) 1932-6203 2015/03 [Refereed][Not invited]
     
    Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression- induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.
  • Seiichiro Ishihara, Motoaki Yasuda, Akihiro Ishizu, Masayori Ishikawa, Hiroki Shirato, Hisashi Haga
    ONCOTARGET 6 (7) 4602 - 4614 1949-2553 2015/03 [Refereed][Not invited]
     
    Radiotherapy is effective for treating various types of tumors. However, some cancer cells survive after irradiation and repopulate tumors with highly malignant phenotypes that correlate with poor prognosis. It is not known how cancer cells survive and generate malignant tumors after irradiation. Here, we show that activating transcription factor 5 (ATF5) promotes radioresistance and malignancy in cancer cells after irradiation. In the G1-S phase of the cell cycle, cancer cells express high levels of ATF5, which promotes cell cycle progression and thereby increases radioresistance. Furthermore, ATF5 increases malignant phenotypes, such as cell growth and invasiveness, in cancer cells in vitro and in vivo. We have identified a new mechanism for the regeneration of highly malignant tumors after irradiation and shown that ATF5 plays a key role in the process.
  • Naoya Yamaguchi, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga
    SCIENTIFIC REPORTS 5 7656  2045-2322 2015/01 [Refereed][Not invited]
     
    Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower'' cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin beta 1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin beta 1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin beta 1, and PI3K are upregulated in leader cells and drive collective cell migration.
  • Sumire Ishida, Ryosuke Tanaka, Naoya Yamaguchi, Genki Ogata, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga
    PLOS ONE 9 (8) e99655  1932-6203 2014/08 [Refereed][Not invited]
     
    Lumen formation is important for morphogenesis; however, an unanswered question is whether it involves the collective migration of epithelial cells. Here, using a collagen gel overlay culture method, we show that Madin-Darby canine kidney cells migrated collectively and formed a luminal structure in a collagen gel. Immediately after the collagen gel overlay, an epithelial sheet folded from the periphery, migrated inwardly, and formed a luminal structure. The inhibition of integrin-beta 1 or Rac1 activity decreased the migration rate of the peripheral cells after the sheets folded. Moreover, lumen formation was perturbed by disruption of apical-basolateral polarity induced by transforming growth factor-beta 1. These results indicate that cell migration and cell polarity play an important role in folding. To further explore epithelial sheet folding, we developed a computer-simulated mechanical model based on the rigidity of the extracellular matrix. It indicated a soft substrate is required for the folding movement.
  • Takeomi Mizutani, Kazuki Takeda, Hisashi Haga, Mitsugu Todo, Kazushige Kawabata
    HISTOCHEMISTRY AND CELL BIOLOGY 141 (5) 473 - 481 0948-6143 2014/05 [Refereed][Not invited]
     
    Simple epitheliums in normal glandular systems are regulated not to stratify even though the constituent cells proliferate and will rise from the epithelium. Since epithelial cells have the potential to establish cell-cell adhesions, the avoidance of stratification must be related to the intracellular signal cascades and the extracellular conditions. The contributions of the former are becoming clarified, but the influence of the latter is poorly understood. In the present study, we examined whether the frequency of cell-on-cell adhesion, which mimics the early stage of multilayering, is dependent on the type of the extracellular scaffold protein. Wild-type epithelial cells were cultured on E-cadherin-Fc (a cell-cell adhesion protein) or collagen (an extracellular matrix protein), and then, green fluorescent protein (GFP)-positive cells were seeded onto these wild-type cells. We observed that the cell-on-cell adhesion (adhesion of the GFP-positive cell to the wild-type cells) was more frequent in the E-cadherin-Fc treatment than the collagen treatment. The cell-on-cell adhesions that were observed in the E-cadherin treatment were transient and decreased in frequency to that of the collagen treatment after the 12 h of cell culture. We observed the disappearance of E-cadherin-Fc but not collagen during cell culture. These results suggest that transient multilayering in simple epithelium is possible, depending on the types of extracellular scaffold protein, and they imply that cells can modify the extracellular conditions to meet normal cellular conditions.
  • Takeomi Mizutani, Kazuki Takeda, Hisashi Haga, Mitsugu Todo, Kazushige Kawabata
    HISTOCHEMISTRY AND CELL BIOLOGY 141 (5) 473 - 481 0948-6143 2014/05 [Refereed][Not invited]
     
    Simple epitheliums in normal glandular systems are regulated not to stratify even though the constituent cells proliferate and will rise from the epithelium. Since epithelial cells have the potential to establish cell-cell adhesions, the avoidance of stratification must be related to the intracellular signal cascades and the extracellular conditions. The contributions of the former are becoming clarified, but the influence of the latter is poorly understood. In the present study, we examined whether the frequency of cell-on-cell adhesion, which mimics the early stage of multilayering, is dependent on the type of the extracellular scaffold protein. Wild-type epithelial cells were cultured on E-cadherin-Fc (a cell-cell adhesion protein) or collagen (an extracellular matrix protein), and then, green fluorescent protein (GFP)-positive cells were seeded onto these wild-type cells. We observed that the cell-on-cell adhesion (adhesion of the GFP-positive cell to the wild-type cells) was more frequent in the E-cadherin-Fc treatment than the collagen treatment. The cell-on-cell adhesions that were observed in the E-cadherin treatment were transient and decreased in frequency to that of the collagen treatment after the 12 h of cell culture. We observed the disappearance of E-cadherin-Fc but not collagen during cell culture. These results suggest that transient multilayering in simple epithelium is possible, depending on the types of extracellular scaffold protein, and they imply that cells can modify the extracellular conditions to meet normal cellular conditions.
  • Takuya Kato, Atsushi Enomoto, Takashi Watanabe, Hisashi Haga, Sumire Ishida, Yuji Kondo, Koichi Furukawa, Takeshi Urano, Shinji Mii, Liang Weng, Maki Ishida-Takagishi, Masato Asai, Naoya Asai, Kozo Kaibuchi, Yoshiki Murakumo, Masahide Takahashi
    CELL REPORTS 7 (4) 1156 - 1167 2211-1247 2014/05 [Refereed][Not invited]
     
    For collective invasion, cancer cells form cohesive groups comprised of leading cells (LCs) at the forefront and following cells (FCs) at the rear. However, the molecular mechanisms that define LCs and FCs remain elusive. Here, we demonstrated that LCs, but not FCs, upregulated the expression of integrin beta 1 after the loss of intercellular adhesion. The LC-specific expression of integrin beta 1 was posttranscriptionally regulated by the TRIM27/MRTF-B complex in response to the loss of intercellular adhesion, thereby regulating the stability and translation of integrin beta 1 mRNA via microRNA-124 in LCs. Accordingly, depletion of TRIM27 and MRTF-B abrogated the upregulation of integrin beta 1 in LCs and blocked the invasion of cancer cell groups in vitro and in vivo. Therefore, our findings revealed that the specific function of LCs was defined by intrinsic mechanisms related to the presence of the cell's free surface, providing insights into the regulation of intratumor heterogeneity.
  • Hiro-taka Masuda, Seiichiro Ishihara, Ichiro Harada, Takeomi Mizutani, Masayori Ishikawa, Kazushige Kawabata, Hisashi Haga
    BIOTECHNIQUES 56 (4) 172 - 179 0736-6205 2014/04 [Refereed][Not invited]
     
    We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.
  • Seiichiro Ishihara, Motoaki Yasuda, Ichiro Harada, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga
    EXPERIMENTAL CELL RESEARCH 319 (19) 2916 - 2927 0014-4827 2013/11 [Refereed][Not invited]
     
    Physical properties of the extracellular matrix (ECM) can control cellular phenotypes via mechanotransduction, which is the process of translation of mechanical stresses into biochemical signals. While current research is clarifying the relationship between mechanotransduction and cytoskeleton or adhesion complexes, the contribution of transcription factors to mechanotransduction is not well understood. The results of this study revealed that the transcription factor NF-kappa B, a major regulator for immunoreaction and cancer progression, is responsive to substrate stiffness. NF-kappa B activation was temporarily induced in H1299 lung adenocarcinoma cells grown on a stiff substrate but not in cells grown on a soft substrate. Although the activation of NF-kappa B was independent of the activity of integrin beta 1, an ECM-binding protein, the activation was dependent on actomyosin contractions induced by phosphorylation of myosin regulatory light chain (MRLC). Additionally, the inhibition of MRLC phosphorylation by Rho kinase inhibitor Y27632 reduced the activity of NF-kappa B. We also observed substrate-specific morphology of the cells, with cells grown on the soft substrate appearing more rounded and cells grown on the stiff substrate appearing more spread out. Inhibiting NF-kappa B activation caused a reversal of these morphologies on both substrates. These results suggest that substrate stiffness regulates NF-kappa B activity via actomyosin contractions, resulting in morphological changes. (C) 2013 Elsevier Inc. All rights reserved.
  • Xue Li, Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Masayori Ishikawa, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga
    PLOS ONE 8 (8) e70905  1932-6203 2013/08 [Refereed][Not invited]
     
    Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as a contributor to the limited benefit of radiotherapy; however, its mechanism is still unclear. We previously showed that subcloned lung adenocarcinoma A549 cells (P cells), which survived 10 Gy IR (IR cells), acquired high invasiveness in vitro. Here, we tried to identify the mechanism by which IR cells increase their invasiveness by examining altered gene expression and signaling pathways in IR cells compared with those in P cells. To simulate the microenvironment in vivo, cells were embedded in a three-dimensional (3D) collagen type I gel, in which the IR cells were elongated, while the P cells were spherical. The integrin expression pattern was surveyed, and expression levels of the integrin alpha 2 and beta 1 subunits were significantly elevated in IR cells. Knockdown of alpha 2 expression or functional blockade of integrin alpha 2 beta 1 resulted in a round morphology of IR cells, and abrogated their invasion in the collagen matrix, suggesting the molecule's essential role in cell spread and invasion in 3D collagen. Epidermal growth factor receptor (EGFR) also presented enhanced expression and activation in IR cells. Treatment with EGFR tyrosine kinase inhibitor, PD168393, decreased the ratio of elongated cells and cell invasiveness. Signaling molecules, including extracellular signal-regulated kinase-1/2 (Erk1/2) and Akt, exhibited higher activation in IR cells. Inhibition of Akt activation by treating with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 decreased IR cell invasion, whereas inhibition of Erk1/2 activation by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 did not. Our results show that integrin alpha 2 beta 1 and EGFR cooperatively promote higher invasiveness of IR-survived lung cancer cells, mediated in part by the PI3K/Akt signaling pathway, and might serve as alternative targets in combination with radiotherapy.
  • Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga
    FEBS LETTERS 587 (6) 732 - 736 0014-5793 2013/03 [Refereed][Not invited]
     
    Radiotherapy is one of the major treatment modalities for malignancies. However, cells surviving irradiation often display high levels of invasiveness. This study shows that irradiation-tolerant lung adenocarcinoma demonstrates high invasive capability depending on dephosphorylation of the myosin regulatory light chain (MRLC). In a collagen gel overlay condition, low-invasive subclones of lung adenocarcinoma (A549P-3) showed a round morphology and diphosphorylation of MRLC. In contrast, irradiation-tolerant A549P-3 cells (A549P-3IR) displayed high invasiveness and a lower level of MRLC diphosphorylation. In addition, inhibition of MRLC phosphatase activity decreased the invasive activity. These findings suggest that A549P-3IR cells acquire high invasiveness through MRLC dephosphorylation. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Naoki Seito, Tadashi Yamashita, Yukinori Tsukuda, Yuichiro Matsui, Atsushi Urita, Tomohiro Onodera, Takeomi Mizutani, Hisashi Haga, Naoki Fujitani, Yasuro Shinohara, Akio Minami, Norimasa Iwasaki
    ARTHRITIS AND RHEUMATISM 64 (8) 2579 - 2588 0004-3591 2012/08 [Refereed][Not invited]
     
    Objective Glycosphingolipids (GSLs) are ubiquitous membrane components that modulate transmembrane signaling and mediate cell-to-cell and cell-to-matrix interactions. GSL expression is decreased in the articular cartilage of humans with osteoarthritis (OA). This study was undertaken to determine the functional role of GSLs in cartilage metabolism related to OA pathogenesis in mice. Methods We generated mice with knockout of the chondrocyte-specific Ugcg gene, which encodes an initial enzyme of major GSL synthesis, using the Cre/loxP system (Col2-Ugcg-/- mice). In vivo OA and in vitro cartilage degradation models were used to evaluate the effect of GSLs on the cartilage degradation process. Results Although Col2-Ugcg-/- mice developed and grew normally, OA changes in these mice were dramatically enhanced with aging, through the overexpression of matrix metalloproteinase 13 and chondrocyte apoptosis, compared to their wild-type (WT) littermates. Col2-Ugcg-/- mice showed more severe instability-induced pathologic OA in vivo and interleukin-1a (IL-1a)induced cartilage degradation in vitro. IL-1a stimulation of chondrocytes from WT mice significantly increased Ugcg messenger RNA expression and up-regulated GSL metabolism. Conclusion Our results indicate that GSL deficiency in mouse chondrocytes enhances the development of OA. However, this deficiency does not affect the development and organization of cartilage tissue in mice at a young age. These findings indicate that GSLs maintain cartilage molecular metabolism and prevent disease progression, although GSLs are not essential for chondrogenesis of progenitor and stem cells and cartilage development in young mice. GSL metabolism in the cartilage is a potential target for developing a novel treatment for OA.
  • Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    ACTA BIOMATERIALIA 7 (10) 3766 - 3772 1742-7061 2011/10 [Refereed][Not invited]
     
    We investigated the dynamics of the cortical cytoskeleton in living cells by analyzing the motion of the endogenous components of the cytoskeleton using scanning probe microscopy (SPM). We performed molecular characterization of the microgranules visualized by SPM in living cells and analyzed the motion of these microgranules via particle tracking. Simultaneous SPM and epifluorescence microscopy observations showed that the microgranules recruited not only actin but also cortactin, which can bind to actin filaments. This indicates condensation of actin filaments at microgranules, leading us to identify them as "cytoskeletal microdomains". High-speed SPM observation and particle-tracking analysis showed that these cytoskeletal microdomains exhibit random walk-like diffusive fluctuations over a timescale of seconds. Inhibition of the molecular motor myosin II, which drives actin filaments, led to subdiffusive fluctuations of the microdomains. These results can be explained by longitudinal sliding of actin filaments stochastically driven by myosin II and the bending motion of the actin filaments in the absence of sliding. Analysis of the cytoskeletal microdomains thus revealed the intrinsic dynamics of the cortical cytoskeleton. (C) 2011 Acts Materialia Inc. Published by Elsevier Ltd. All rights reserved.
  • Takeshi Nishioka, Motoaki Yasuda, Tsuguhide Takeshima, Hisashi Haga, Yusuke Miyai, Ken-ichiro Shibata, Rie Yamazaki, Hiroki Shirato, Masahiro Teduka, Hiroyuki Date
    CELL STRUCTURE AND FUNCTION 36 (1) 13 - 20 0386-7196 2011 [Refereed][Not invited]
     
    Purpose: Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation". To better clarify the mechanism, gene expression profiling and pathological experiments were performed. Materials and Methods: A mouse fibrosarcoma cell line, QRsP, was used. Cells were irradiated with 10 Gy. Colony assay and cloning were performed. Six clones were established. cDNA analysis was performed on the clone that showed the largest number of colonies on the 2nd 10 Gy irradiation. Mouse transplantation experiment was then carried out. Results: cDNA analysis showed that cyclin-dependent kinase inhibitors, p16 and p57 were down-regulated; 14.8- and 12.0-fold, respectively for the tolerant clone. Matrix metalloproteinase 3 and 13 were up-regulated; 22.5- and 25.8-fold, respectively. Transplantation ratio was 100% (5/5) for the tolerant clone whereas it was 40% (2/5) for the parent. Under light microscope, the mean mitotic cell number was 4.0+/-3.9 for the parent, and 12.8+/-3.4 for the tolerant clone (p < 0.01, Student's t-test). Conclusions: This study implies that repopulation is not a temporary reaction to irradiation. It is caused probably by "clonal" gene-expression changes, though it remains unknown whether the changes are attributable to tolerant cell selection or to gene mutation/modification.
  • Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 403 (3-4) 363 - 367 0006-291X 2010/12 [Refereed][Not invited]
     
    Filamentous actin and myosin-II are major determinants of cell mechanics and are tightly regulated by a small guanosine triphosphatase, RhoA, and its downstream effectors. We examined the effects of constitutively active mutants of RhoA effectors, which have not been reported before, on cortical stiffness of living cells by using scanning probe microscopy, fluorescence microscopy, and truncated mutants of RhoA effectors labeled with a fluorescent protein. Our data indicated that expression of a constitutively active mutant of Dial, a formin-family actin polymerizer, enhanced cortical stiffness and increased actin filament quantity in cells. Furthermore, expression of a constitutively active mutant of Rho-associated coiled-coil kinase, a myosin-II activator, softened the cell cortex but increased myosin-II activity. Our findings provide new insights into anomalous mechanics of cells, which is a topic of current interest in a variety of biological research fields. (C) 2010 Elsevier Inc. All rights reserved.
  • Hiromasa Suzuki, Huda Muhammad Nurul, Takahiro Seki, Taisuke Kawamoto, Hisashi Haga, Kazushige Kawabata, Yukikazu Takeoka
    MACROMOLECULES 43 (23) 9945 - 9956 0024-9297 2010/12 [Not refereed][Not invited]
     
    A high-density polymer brush of poly(N-isopropylacrylamide) (PNIPA) was precisely prepared following carefully selected procedures, which included selecting the underlying substrate, preparing its surface, and grafting PNIPA on the substrate. As a result, the graft density and the dried thickness of the brush reached more than 0.5 chains/nm(2) and 200 nm, respectively, for the best combination of each procedure. This high-density polymer brush showed gradual collapse with increasing temperature in water, which must be attributed to both the low swelling and the low shrinking abilities of the brush that result from the physically constrained state of the polymers. The contact angle of the air bubbles underneath the high-density polymer brush also gradually decreased up to around 25 degrees C in water with increasing temperature, which indicates that the hydrophilicity of the surface decreases as it does in typical PNIPA-grafted membranes and gels. Starting at the lower critical solution temperature of free PNIPA in water, approximately 32 degrees C, the value of the contact angle started to increase dramatically, and it became constant when the solution temperature exceeded 40 degrees C. Ultimately, the surface exhibited a mostly hydrophilic nature at higher temperatures. The temperature-dependent contact angles can be interpreted by assuming that the terminally chlorinated alkyl groups of the elongated PNIPAs can be positioned on the surfaces or hidden in the vicinity of the membranes, depending on the temperature of the solution.
  • Kensuke Ikeda, Takeomi Mizutani, Osamu Hoshi, Tatsuo Ushiki, Hisashi Haga, Kazushige Kawabata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 400 (1) 181 - 186 0006-291X 2010/09 [Not refereed][Not invited]
     
    The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (similar to 1 mm) into chromosomes (similar to 1 mu m). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) II alpha, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes. (C) 2010 Elsevier Inc. All rights reserved.
  • Kensuke Ikeda, Takeomi Mizutani, Osamu Hoshi, Tatsuo Ushiki, Hisashi Haga, Kazushige Kawabata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 400 (1) 181 - 186 0006-291X 2010/09 [Refereed][Not invited]
     
    The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (similar to 1 mm) into chromosomes (similar to 1 mu m). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) II alpha, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes. (C) 2010 Elsevier Inc. All rights reserved.
  • Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Takeshi Nishioka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 396 (3) 651 - 655 0006-291X 2010/06 [Refereed][Not invited]
     
    Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta 1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta 1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta 1-dependent phenotype, and integrin beta 1 might be a potentially effective therapeutic target in combination with radiotherapy. (C) 2010 Elsevier Inc. All rights reserved.
  • R. Mimura, T. Kamishima, K.C. Kubota, F. Nakano, I. Yabe, H. Sasaki, S. Maruyama, N. Shinohara, A.A. Harris, H. Haga, H. Shirato, S. Terae
    Japanese Journal of Radiology 28 (4) 309 - 313 1867-1071 2010/05 [Refereed][Not invited]
  • Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    HISTOCHEMISTRY AND CELL BIOLOGY 133 (1) 59 - 67 0948-6143 2010/01 [Refereed][Not invited]
     
    In this study, we aimed at improving the temporal resolution of scanning probe microscopy (SPM) for observing living cells by introducing soft cantilevers, low feedback-gain operations, and cantilever deflection imaging. We achieved visualization of the mechanical architecture in leading lamellae of living fibroblasts at a temporal resolution of around 10 s, which is higher than that of conventional contact-mode SPM. Time-lapse SPM could be used to monitor not only cytoskeletal dynamics but also the dynamics of numerous microgranules. Statistical analysis of microgranular motion revealed that the microgranules have superdiffusive behaviors and significant directional order of motion. We also found that the direction of their motion is correlated with the direction of growing actin stress fibers. The combination of SPM with fluorescence microscopy showed that vinculin, a component of cell-substratum adhesion sites, localizes at the microgranules. Our experimental data provides a new insight into the intracellular mechanical architecture and its structural dynamics, suggesting that high-speed live-cell SPM has great potential for investigating the structural origin of cellular dynamics.
  • ISHIHARA Seiichiro, HAGA Hisashi, YASUDA Motoaki, MIZUTANI Takeomi, KAWABATA Kazushige, SHIRATO Hiroki, NISHIOKA Takeshi
    Biochemical and Biophysical Research Communications 396 (3) 651 - 655 0006-291X 2010 [Not refereed][Not invited]
  • Waka Mitsui, Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 72 (4-5) 227 - 234 0914-9465 2009/12 [Refereed][Not invited]
     
    The mechanical memory effect of single cells was reported in our recent study. In order to clarify this effect, various sequential stimuli of uniaxial deformation were applied to cells by deformable culture dishes and a deformation device, and the local stiffness distribution of single C2C12 myoblasts was visualized by scanning probe microscopy. Following a single step stretching, cellular stiffness first increased steeply and then gradually decreased for two hours. By a single step stretching 30 min after a long pulse-like deformation with a pulse duration of 30 min, the cells responded in the same way. On the other hand, they (lid not respond to a single step stretching 39 min after a short pulse-like deformation with a pulse duration of 0.5 min. These results indicated that cellular mechanical response to external deformation is affected strongly by a preceding deformation and that the duration time of the preceding deformation is an important factor in the change in mechanical response. We consider that the change in mechanical response contributes to a regulatory mechanism of cellular contractile force.
  • Takeomi Mizutani, Hisashi Haga, Kosaku Kato, Kazushige Kawabata
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 72 (4-5) 235 - 243 0914-9465 2009/12 [Refereed][Not invited]
     
    Scanning probe microscopy (SPM) provides a range of strategies for studying biological phenomena due to its ability to image surfaces under liquids. However, some cellular events, such as cell migration, exceed the maximum measurable range of SPM. Recently, we have developed a wide range scanning probe microscope (WR-SPM) to investigate cellular events which exceed the range of the conventional SPM. In this review, we introduce the instrumentation of the WR-SPM, which can measure a sample for 400 mu m in the xy directions and 23 mu m in the z direction. We then show the application of the WR-SPM to studies of the stiffness response of epithelial cells to an external loading force and demonstrat that the stiffness of the epithelial cells increases under stretched conditions. We also showed the results on the mesh structure on the surface of a melanoma cell as well as the regulatory mechanism of the cellular contractile force by the combined use of topographical and mechanical modes of the WR-SPM. These findings indicate that the WR-SPM is very useful for studying the functions of a cell in relation to the surface structure and mechanical properties of that cell.
  • Takeomi Mizutani, Kazushige Kawabata, Yoshikazu Koyama, Masayuki Takahashi, Hisashi Haga
    CELL MOTILITY AND THE CYTOSKELETON 66 (7) 389 - 397 0886-1544 2009/07 [Refereed][Not invited]
     
    Fibroblasts regulate their contractile force in response to external stretch; however, the detailed mechanism by which the force is regulated is unclear. Here, we show that diphosphorylation and dephosphorylation of myosin regulatory light chain (MRLC) are involved in the stretch-induced force response. Cellular stiffness, which reflects the cellular contractile force, under external stretch was measured by mechanical-scanning probe microscopy. Fibroblasts (NIH-3T3) expressing green fluorescent protein (GFP)-tagged mutant-type MRLC (MRLC(T18A)-GFP), which cannot be diphosphorylated, did not show any stretch-induced stiffness response, whereas the stiffness in cells expressing GFP-tagged wild-type MRLC(MRLC(WT)-GFP) increased immediately after the stretch and subsequently decreased after 1-2 h. Urea-PAGE western blot analysis showed that the proportion of diphosphorylated MRLC (PP-MRLC) transiently increased after the stretch and decreased after 1-2 h. Dominant-negative RhoA (RhoA(N19))-expressing cells did not show the stiffness response to the stretch, whereas wild-type RhoA-expressing cells did. It was concluded that file Cellular force response originates in the stretch-induced diphosphorylation and dephosphorylation of MRLC and is regulated via the RhoA signaling cascade Cell Motil. Cytoskeleton 66: 389-397,2009. (C) 2009 Wiley-Liss, Inc.
  • Intracellular Particles Involved in Stress Fiber Formation through Remodeling of Actin Filament Networks
    Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    BIOPHYSICAL JOURNAL 96 (3) 123A - 123A 0006-3495 2009/02 [Refereed][Not invited]
  • Kaori Tsutsumi, Masumi Tsuda, Natsuka Yazawa, Hirotaka Nakamura, Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Rie Yamazaki, Hiroki Shirato, Hideaki Kawaguchi, Takeshi Nishioka, Yusuke Ohba
    CELL STRUCTURE AND FUNCTION 34 (2) 89 - 96 0386-7196 2009 [Refereed][Not invited]
     
    Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta 1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.
  • Takeshi Nishioka, Yusuke Miyai, Hisashi Haga, Kazushige Kawabata, Hiroki Shirato, Akihiro Homma, Kenichiro Shibata, Motoaki Yasuda
    CELL STRUCTURE AND FUNCTION 34 (1) 17 - 22 0386-7196 2009 [Refereed][Not invited]
     
    Purpose: To find a new molecule that affects p53-dependent radiosensitivity.Methods and Materials: A mouse sarcoma cell line, QRsP(p53+/+), was used. From this cell line, we established a radiosensitive clone and a radioresistant one. Colony assay, p53 gene transfer, a luciferase assay for p53 and p21, animal transplantation experiment, and DNA array analyses were performed.Results: Microarray showed marked reduction of a transcription factor, ATF5, both in vitro and in vivo for the radiosensitive clone. Interestingly, flow cytometric analysis demonstrated marked apoptosis for the radiosensitive clone by p53 cloned adenovirus infection. Luciferase reporter assay revealed that ATF5 suppressed the transactivational activity of p53 and p63. By ATF5 gene transfer, the radiosensitive clone regained resistance to both ionizing-radiation and Ad-p53 infection-induced cell death. Surprisingly, time-lapse cell migration observation revealed greater cell motility for ATF5-transfected radiosensitive clone.Conclusions: It seems likely that ATF5 is a potent repressor of p53 and elevated expression of ATF5 in a tumor may relate to enhanced malignant phenotypes, such as radioresistance or greater cell motility.
  • Taisuke Kawamoto, Hisashi Haga, Kazushi Tamura, Takeomi Mizutani, Kazushige Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS 47 (7) 6173 - 6176 0021-4922 2008/07 [Not refereed][Not invited]
     
    Mechanical stimuli such as cyclic stretch and fluid stress affect various cellular physiologies, including proliferation. morphology. and differentiation. We investigated Cellular response to shrinking stimuli by developing an isotropic deformation device and observing cellular elasticity with mechanical scanning probe microscopy (M-SPM). The isotropic deformation device consists of a steel ring and a deformable elastic culture dish made of transparent silicone rubber. The M-SPM can visualize topography and spatial distribution of local elasticity of biomaterials in solution. Fibroblasts became softer in response to 6% shrinkage. Cell elasticity did not increase for 1 h after the shrinking stimulus. Inhibitory studies using lysophosphatidic acid and calyculin-A revealed that myosin light chain phosphatase leading to dephospholylation of myosin II regulatory light chain is involved in cell softening.
  • Nishioka T, Yasuda M, Tsutsumi K, Haga H, Shirato H
    Radiation medicine 25 (8) 430 - 431 1862-5274 2007/10 [Refereed][Not invited]
  • Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 46 (8B) 5631 - 5635 0021-4922 2007/08 [Not refereed][Not invited]
     
    This study was aimed at developing a novel experimental setup to investigate the tensional response of a single living cell to external deformation. We constructed the setup which consisted of three components: a device for applying various patterns of uniaxial deformation to living cells, a mechanical scanning probe microscope (M-SPM), and an optical microscope. Intracellular tension is reflected by cellular stiffness measured by M-SPM. Using the setup, we found that stiffness in some areas on a living fibroblast cell increased while stiffness in other areas decreased under 8% external single-step stretch. Furthermore, we found that repetitive deformation inhibited the increase in the stretch-induced cellular stiffness. The present setup is useful for investigating the characteristics of, intracellular tensional responses to external deformation.
  • Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata
    ACTA BIOMATERIALIA 3 (4) 485 - 493 1742-7061 2007/07 [Refereed][Not invited]
     
    We have developed a new stretch device to investigate the biomechanical responses to an external loading force on a tissue-like material consisting of cells and a collagen gel. Collagen gel, a typical matrix found abundantly in the connective tissue, was attached to an elastic chamber that was precoated with a thin layer of collagen. Madin-Darby canine kidney cells that were cultured on the collagen gel were stretched in a uniaxial direction via deformation of the elastic chamber. Changes in the morphology and stiffness of the tissue-like structure were measured before and after the stretch using wide-range scanning probe microscopy (WR-SPM). The change in cellular morphology was heterogeneous, and there was a twofold increase in the intercellular junction due to the stretch. In addition to the WR-SPM measurements, this device enables observation of the spatial distribution of cytoskeletal proteins such as vimentin and alpha-catenin using immunofluorescent microscopy. We concluded that the stretch device we have reported in this paper is useful for measuring the mechanical response of a tissue-like material over a range of cell sizes when exposed to an external loading force. (c) 2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
  • Hisashi Haga, Masafumi Nagayama, Kazushige Kawabata
    CURRENT NANOSCIENCE 3 (1) 97 - 103 1573-4137 2007/02 [Not refereed][Not invited]
     
    Scanning probe microscope (SPM) has been developed as a powerful tool for obtaining high resolution topographic images of biological samples in their natural aqueous environment. SPM can also be used to evaluate mechanical properties because its probe is physically in contact with the samples during measurement. To obtain cellular stiffness with SPM, we have proposed two methods: a force modulation mode and a force snapping mode. Considering the influence of the drag force of liquids, we have successfully improved the quantitative evaluation of cellular stiffness by using the force modulation mode. Experiments performed using the two methods revealed that the local stiffness of fibroblasts was not homogeneous on the cell surface but largely varied from point to point. It was revealed that spatial and temporal distributions of cellular stiffness originate in cytoskeletal distribution, mode of cellular migration, and intracellular contractile force.
  • Novel beads-and-chain structure of unraveling human chromosomes by mechanical stretching
    Kensuke Ikeda, Osamu Hoshi, Tatsuo Ushiki, Hisashi Haga, Kazushige Kawabata
    BIOPHYSICAL JOURNAL 52A - 52A 0006-3495 2007/01 [Refereed][Not invited]
  • Three-dimensional tumor cell movement in collagen gel: mesenchymal-to-amoeboid transition in a sub-clone that survived 10 Gy irradiation
    T. Nishioka, H. Haga, Y. Miyai, M. Yasuda, K. Tsutsumi, R. Yamazaki, H. Shirato, K. Kawabata
    INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS 69 (3) S148 - S148 0360-3016 2007 [Not refereed][Not invited]
  • Collective Movement and Morphogenesis of Epithelial Cells
    Proceedings of the international symposium on topological aspects of critical systems and networks 82 - 85 2007 [Not refereed][Not invited]
  • Takeomi Mizutani, Hisashi Haga, Yoshikazu Koyama, Masayuki Takahashi, Kazushige Kawabata
    JOURNAL OF CELLULAR PHYSIOLOGY 209 (3) 726 - 731 0021-9541 2006/12 [Refereed][Not invited]
     
    Regulation of the contractile force is crucial for cell migration, cell proliferation, and maintenance of cell morphology. Phosphorylation of the myosin II regulatory light chain (MRLC) is involved in these processes. To show whether the diphosphorylation of MRLC increases the tension acting on stress fibers, changes in the stiffness of fibroblasts expressing wild-type MRLC and a mutant type, which cannot be diphosphorylated, on treatment with lysophosphatidic acid (LPA) were examined by a mechanical-scanning probe microscope (M-SPM). The LPA treatment increased cellular stiffness in the wild-type MRLC expressing cells, while it had no effect on the mutated cells. immunostaining showed that LPA stimulation induced the diphosphorylation of MRLC. These results suggest that the diphosphorylation of MRLC enhances the tension acting on stress fibers.
  • K Kato, Y Ohmori, T Mizutani, H Haga, K Ohashi, T Ito, K Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 45 (3B) 2328 - 2332 0021-4922 2006/03 [Not refereed][Not invited]
     
    The role of filamin A (FLNa) in the organization of stress fibers has been studied by comparing the mechanical properties of FLNa-deficient human melanoma cells (M2 cells) and M2 sub-line expressing FLNa (A7 cells). We measured both the topographies and the elasticity distributions of M2 and A7 cells by using a wide-range scanning probe microscopy. In A7 cells, we observed aligned fibrous structures, whereas in M2 cells, fibrous structures were dispersed randomly. Immunofluorescent observation revealed that the aligned fibrous structures in A7 cells were stress fibers generating intracellular tension. On the other hand, the reticular structures observed in M2 cells did not correspond to actin filaments. The cellular stiffness of A7 cells was approximately twice than that of M2 cells, indicating that A7 cells produce larger contractile force through the stress fibers. These results suggest that FLNa stabilizes the stress fibers and increases the cellular stiffness.
  • T Mizutani, H Haga, K Kato, K Matsuda, K Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 45 (3B) 2353 - 2356 0021-4922 2006/03 [Not refereed][Not invited]
     
    Spatial distributions for 100-mu m(2)-stiffness of type I collagen gels were measured by wide-range scanning probe microscopy. A cantilever being attached to the tip of a glass bead of 100 mu m diameter was used to reduce local stress during Measurements. This method enabled the cantilever to be ill contact With the gel Surface in it manner of it Hertzian contact model regardless of the rough meshwork formation of collagen gels. Stiffness images of collagen gets showed stiff domain structures With it size of 20 mu m. The more the concentration of collagen increased, the more the stiffness increased with the growth of stiff domain structures. Immunostaining for collagen molecules showed highly concentrated fibril structures as large as the stiff domain structures. These results indicate that the structure of collagen gel is nonuniform in the range of 100 mu m square, but it is heterogeneous because of collagen fibril aggregation.
  • T Nitta, H Kato, H Haga, K Nemoto, K Kawabata
    JOURNAL OF THE PHYSICAL SOCIETY OF JAPAN 74 (11) 2875 - 2879 0031-9015 2005/11 [Not refereed][Not invited]
     
    We measure the static friction F-c of agar gels on glass substrates immersed in water. F-c is independent of the nominal contact area, and increases with the normal load and the duration t(w) of contact prior to sliding. Using a confocal laser-scanning microscope, many fine dark spots are clearly visible in the optical reflection distribution images on the glass interface in contact with the gel. The total area of the dark spots increases with t(w), corresponding qualitatively to that of F-c. These observations indicate that F-c originates from the formation of the contacting spots at the interface, which is not due to asperities of the gel surface alone but related to the drainage of water trapped by the gel polymer network at the frictional interface.
  • K Nomura, O Hoshi, D Fukushi, T Ushiki, H Haga, K Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 (7B) 5421 - 5424 0021-4922 2005/07 [Not refereed][Not invited]
     
    We succeeded in visualizing the spatial distribution of the local elasticity of mitotic human chromosomes in a liquid environment using scanning probe, microscopy (SPM). Force-versus-indentation curves (force curves) were collected over an entire single chromosome. To estimate the local elasticity of thin chromosomes from the force curves, we examined the validity of a previously proposed model that takes into account the effect of the finite thickness of samples on the estimation of the local elasticity. The force curves obtained are well represented by the model within a small indentation range. The elasticity obtained is independent of the indentation within an indentation range of 100nm. Such fitting procedures for the force curves collected are carried out over the entire chromosome, and the elasticity distribution of a single chromosome is visualized.
  • S Toko, T Mizutani, H Haga, K Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 (7B) 5451 - 5454 0021-4922 2005/07 [Not refereed][Not invited]
     
    The spatiotemporal variation in the local stiffness of fibroblasts was visualized successfully using wide-range scanning probe microscopy (SPM) in the force mapping mode when the cells attach and spread on a substratum, to clarify the cellular mechanical effects on the development of morphological polarity. We found that the stiffness distribution in inhomogeneous in a circular cell. When the cell shape is polarized, several stiff bands appear clearly along the direction of the morphological polarity. The SPM measurements give new information on the cellular mechanical effect on the development of morphological polarity.
  • H Haga, C Irahara, R Kobayashi, T Nakagaki, K Kawabata
    BIOPHYSICAL JOURNAL 88 (3) 2250 - 2256 0006-3495 2005/03 [Refereed][Not invited]
     
    Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, the directions of cell movement gradually converged on one direction as the number of cells increased, whereas the cells moved randomly on the glass substrate. We also observed "leader'' cells, which extended large lamellae and were accompanied by many "follower'' cells, migrating in the direction of oriented collagen fibers. The mean-squared displacement of each cell movement and the spatial correlation function calculated from the spatial distribution of cell velocity were obtained as functions of observation time. In the case of the gel substrate, the spatial correlation length increased gradually, representing the collectiveness of multicellular movement.
  • T Mizutani, H Haga, K Kawabata
    CELL MOTILITY AND THE CYTOSKELETON 59 (4) 242 - 248 0886-1544 2004/12 [Refereed][Not invited]
     
    Stiffness responses of fibroblasts were measured by scanning probe microscopy, following elongation or compression by deformation of an elastic substrate by 8%. The cellular stiffness, reflecting intracellular tension acting along stress fibers, decreased or increased instantly in response to the elongating or compressing stimuli, respectively. After this rapid change, the fibroblasts gradually recovered to their initial stiffness during the following 2 h, and then stabilized. The cells did not show conspicuous changes in shape after the 8% deformation during the SPM measurements. Fluorescence examination for GFP-actin demonstrated that the structure of the stress fibers was not altered noticeably by this small degree of deformation. Treatment with Y-27632, to inhibit myosin phosphorylation and abrogate cellular contractility, eliminated the change in stiffness after the mechanical elongation. These results indicate that fibroblasts possess a mechanism that regulates intracellular tension along stress fibers to maintain the cellular stiffness in a constant equilibrium state. (C) 2004 Wiley-Liss, Inc.
  • M Nagayama, H Haga, M Takahashi, T Saitoh, K Kawabata
    EXPERIMENTAL CELL RESEARCH 300 (2) 396 - 405 0014-4827 2004/11 [Refereed][Not invited]
     
    Scanning probe microscopy and immunofluorescence observations indicated that cellular stiffness was attributed to a contractile network structure consisting of stress fibers. We measured temporal variations in cellular stiffness when cellular contractility was regulated by dosing with lysophosphatidic acid or Y-27632. This experiment revealed a clear relation between cellular stiffness and contractility: Increases in contractility caused cells to stiffen. On the other hand, decreases in contractility reduced cellular stiffness. In both cases, not only the stiffness of the stress fibers but also that of the whole of the cell varied. Immunofluorescence observations of myosin II and vinculin indicated that the stiffness variations induced by the regulation of cellular contractility were mainly due to rearrangements of the contractile actin network on the dorsal surface. Taken together, our findings provide evidence that the actin cytoskeletal network and its contractility features provide and modulate the mechanical stability of adherent cells. (C) 2004 Elsevier Inc. All rights reserved.
  • T Mizutani, H Haga, K Nemoto, K Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS SHORT NOTES & REVIEW PAPERS 43 (7B) 4525 - 4528 0021-4922 2004/07 [Not refereed][Not invited]
     
    We developed wide-range scanning probe microscope (WR-SPM) for visualizing the topography and mechanical properties of samples in the submillimeter range. A piezoelectric scanner with a maximum xy scan range of 400 pin square and a z range of 16 pin was constructed by joining two commercial piezotranslators in tandem. The reliability of measurement was confirmed by measuring a well-defined pattern engraved on a silicon substrate. Using the WR-SPM, the spatial distribution of stiffness and time-lapse images of a large colony consisting of approximately 40 epithelial cells were visualized Successfully, where the local stiffness was measured using the force mapping mode. These results indicate that the WR-SPM can be a powerful instrument to visualize the mechanical properties of biological phenomena extending in the submillimeter range.
  • Visualization of dynamic organization of cytoskeleton gels in living cells by hybrid-SPM
    K Kawabata, Y Sado, M Nagayama, T Nitta, K Nemoto, Y Koyama, H Haga
    CHINESE JOURNAL OF POLYMER SCIENCE 21 (2) 169 - 174 0256-7679 2003/03 [Not refereed][Not invited]
     
    We succeeded in performing of hybrid Scanning Probe Microscopy (hybrid-SPM) in which mechanical-SPM and fluorescence microscopy are combined. This technique is able to measure simultaneously mechanical properties and distribution of cytoskeletons of living cells by using green fluorescent protein. We measured evolution of both local elasticity and distributions of actin stress fibers in an identical fibroblast living in physiological conditions. The SPM experiments revealed that stiffer lines develop in living cells, which correspond to actin stress fibers. The elasticity of the actin stress fibers is as high as 100 kPa. We discuss mechanical effects on the development of actin filament networks.
  • T Nitta, Y Endo, H Haga, K Kawabata
    JOURNAL OF ELECTRON MICROSCOPY 52 (3) 277 - 281 0022-0744 2003 [Refereed][Not invited]
     
    The inhomogeneous structure of agar gels was examined by means of mechanical-scanning probe microscopy Several domains were observed in the elasticity images, while such domains could not be seen in the height images. The domain size decreased with increases in agar concentration. We found that the histograms of the logarithm of the local elastic modulus were described well by a single normal distribution. As the agar concentration increased, the peak values of the histograms increased, while the half-value width remained constant. These results imply that the gelation process of agar gels has a common mechanism, despite its complexity.
  • T Nitta, H Haga, K Kawabata
    JOURNAL DE PHYSIQUE IV 12 (PR9) 319 - 320 1155-4339 2002/11 [Not refereed][Not invited]
     
    We measured the static friction force of agar gel-on-glass plate ill water The static friction force is independent of the apparent contact area between the agar gel and file glass plate. It increases with waiting time, that is, contact duration prior to motion. The static friction force is represented well by a power law of waiting time. The waiting time dependence is different from those of solid-on-solid systems. These results arc discussed, based oil asperity contact model.
  • M Nagayama, H Haga, Y Tanaka, Y Hirai, M Kabuto, K Kawabata
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS SHORT NOTES & REVIEW PAPERS 41 (7B) 4952 - 4955 0021-4922 2002/07 [Not refereed][Not invited]
     
    We improved the force modulation mode with scanning probe microscopy (SPM) in order to make a quantitative evaluation of the viscoelasticity of living cells. Taking account of the viscosity of liquid medium, the vibration frequency of the cantilever was selected to be 500 Hz, and analysis of cantilever vibration was adopted for evaluation of the viscoelasticity of the samples. Consequently, we have succeeded in determining viscoelasticity distribution on living cells. The values of Young's modulus and the coefficient of viscosity vary from 10 to 50 kPa and from 20 to 40 Pa(.)s on a cell, depending on its internal cellular structure.
  • M Nagayama, H Haga, K Kawabata
    CELL MOTILITY AND THE CYTOSKELETON 50 (4) 173 - 179 0886-1544 2001/12 [Not refereed][Not invited]
     
    Sequential images of the local stiffness distribution of living fibroblasts (NIH3T3) were captured under a culture condition using scanning probe microscopy in a force modulation mode. We found a clear relation between cell mi-ration and local stiffness distribution on the cell: When cells were stationary at one position, the stiffness distribution of their cellular surface was quite stable. On the other hand, once the cells started to move, the stiffness in their nuclear regions drastically decreased. Possible explanations for the correlation between the cell mi-ration and the cell stiffness are proposed. Cell Motil. Cytoskeleton 50: 173-179,2001. (C) 2001 Wiley-Liss, Inc.
  • T Saitoh, S Takemura, K Ueda, H Hosoya, M Nagayama, H Haga, K Kawabata, A Yamagishi, M Takahashi
    FEBS LETTERS 509 (3) 365 - 369 0014-5793 2001/12 [Not refereed][Not invited]
     
    We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin HA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin HA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin HA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
  • Kazushige Kawabata, Masafumi Nagayama, Hisashi Haga, Takashi Sambongi
    CURRENT APPLIED PHYSICS 1 (1) 66 - 71 1567-1739 2001/01 [Not refereed][Not invited]
     
    Animal cells crawl in tissue to achieve their functions. The cell locomotion is a typical case of collective phenomena in biological systems. By atomic force microscopy (AFM), we measured the spatial distribution of local elasticity over cells of fibroblasts, which are kept alive in physiological conditions. The AFM experiments revealed that: (1) the nucleus area of the cell surface is about 10 times softer than the surroundings, and (2) distribution of local elasticity is evolving in time. The drug-dosing experiments confirm that the cell shape and stiffness originate mainly in actin filaments but not in microtubules. The elasticity pattern does not always correspond to that of actin filaments from fluorescence observations. These results indicate that the cell stiffness results not only from the density of actin filaments but also from their dynamical properties. (c) 2001 Elsevier Science B.V. All rights reserved.
  • H Shiga, Y Yamane, E Ito, K Abe, K Kawabata, H Haga
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS SHORT NOTES & REVIEW PAPERS 39 (6B) 3711 - 3716 0021-4922 2000/06 [Not refereed][Not invited]
     
    In order to examine the mechanical properties of the membrane surface of astrocytes, we observed living astrocytes by atomic force microscopy (AFM) both in contact mode and force-mapping mode. Ridge-like structures reflecting actin filaments were observed in the topographic images in contact mode, but not in force-mapping mode, using a zero-loading force. When we measured the elasticity of astrocytes, we observed that the cell membrane above the nucleus was soft and the cell membrane above the cytosol was stiff. In particular, the parts reflecting actin filaments were very stiff. This effect of actin filaments on the elasticity of astrocytes was confirmed by the loss of actin filaments after application of actin-polymerization inhibitor.
  • H Haga, S Sasaki, K Kawabata, E Ito, T Ushiki, T Sambongi
    ULTRAMICROSCOPY 82 (1-4) 253 - 258 0304-3991 2000/02 [Not refereed][Not invited]
     
    Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments - actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity. (C) 2000 Elsevier Science B.V. All rights reserved.
  • T Nitta, H Haga, K Kawabata, K Abe, T Sambongi
    ULTRAMICROSCOPY 82 (1-4) 223 - 226 0304-3991 2000/02 [Not refereed][Not invited]
     
    Measurements of the local elastic modulus of agar gels obtained with atomic force microscope (AFM) force mapping were compared with values obtained by the tensile creep method. The observed spatial distributions of the local elastic modulus over the gel surface in AFM elastic images clearly corresponded to the network structure of agar fibers observed both in AFM topographic and scanning electron microscope (SEM) images. Both peak and average values of distribution functions in the histograms of local elastic modulus increase monotonically with the agar concentration. Values obtained by AFM force mapping were found to be proportional to values obtained from creep experiments. (C) 2000 Elsevier Science B.V. All rights reserved.
  • T Tojima, Y Yamane, H Takagi, T Takeshita, T Sugiyama, H Haga, K Kawabata, T Ushiki, K Abe, T Yoshioka, E Ito
    NEUROSCIENCE 101 (2) 471 - 481 0306-4522 2000 [Not refereed][Not invited]
     
    We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100 nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells. (C) 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.
  • Quantitative analyses of topography and elasticity of living and fixed astrocytes
    Y Yamane, H Shiga, H Haga, K Kawabata, K Abe, E Ito
    JOURNAL OF ELECTRON MICROSCOPY 49 (3) 463 - 471 0022-0744 2000 [Not refereed][Not invited]
     
    The topography and elasticity of living and fixed astrocytes cultured from the rat cerebra were studied quantitatively by atomic force microscopy (AFM). Ridge-like structures reflecting F-actin beneath the cell membrane were prominent in the contact-mode images of living astrocytes. Many of these ridges became unclear after fixation (2%, glutaraldehyde). In addition, the ridge-like structures were invisible in the topography of living cells observed at zero-loading force in the force mapping mode, which is considered to show the real cell surface not pressed down by an AFM tip. The topography of fixed cells observed both in the contact mode and at zero-loading force in the force mapping mode was similar to that of living cells observed at zero-loading force in the force mapping mode, although some deformed areas were detected in the fixed cells. The elasticity map images of living astrocytes showed that the cell membrane above the nucleus was softer (2-3 kPa) than the surroundings, and that the cell membrane above F-actin was stiffer (10-20 kPa) than the surroundings. In the elasticity map images of fixed astrocytes, on the other hand, the elasticity of the cells was found to be relatively uniform (200-700 kPa) irrespective of the inner structures of cells. These results show that images observed by AFM should be carefully examined in consideration of the force introduced to specimens and the elasticity of specimens to find out the real surface topography.
  • Time-lapse viscoelastic imaging of living fibroblasts using force modulation mode in AFM
    H Haga, M Nagayama, K Kawabata, E Ito, T Ushiki, T Sambongi
    JOURNAL OF ELECTRON MICROSCOPY 49 (3) 473 - 481 0022-0744 2000 [Not refereed][Not invited]
     
    Using the force modulation mode in atomic force microscopy, we have succeeded in capturing time-lapse viscoelastic images of living mouse fibroblasts (NIH3T3) for several hours in a physiological condition without damaging the fibroblasts. Elongation of the lamellipodia and swelling of blabs were observed in time-lapse topographic images, which were taken every 10 min. The corresponding viscoelastic responses at a frequency of 600 Hz were visualized as consecutive images. The stiffer part of the cell body was fairly stable and did not show morphological changes for over 1 h. This is probably due to excess condensation of the actin network, hardening the cell cortex, and lowering the cytoskeletal activity. The nuclear portion of the cell body seems to be slightly less viscous than the peripheral region.
  • Big softer hole on living cells: Elasticity imaging with AFM
    K Kawabata, H Haga, T Nitta, Y Endo, M Nagayama, E Ito, T Sambongi
    SCANNING AND FORCE MICROSCOPIES FOR BIOMEDICAL APPLICATIONS II 3922 91 - 98 0277-786X 2000 [Not refereed][Not invited]
     
    We have focused on effects of local mechanical properties of the cell on cell motion. By using atomic force microscopy, we measured spatial distribution of local elastic modulus on mouse fibroblasts (NIH3T3), which is living in a physiological condition. In order to examine validity of AFM elastic measurements, we measured local elastic modulus of gels as elastic reference materials. The results obtained with AFM were compared with values obtained by tensile creep method. It is veryfied that these values are proportional each other. The AFM experiments on living cells revealed that center area of cell surface is about 10 times softer than the surroundings and looks like a big softer hole in the elasticity image. We fixed the cell just after the AFM measurements and carried out immunofluorescence observation for cytoskeletal filaments of actin filaments, microtubules and intermediate filaments. A comparison between distribution of local elasticity and cytoskeletones indicates that harder area on the cell results mainly from concentration of actin filaments. However, We found that some areas like the big softer hole do not correspond to distribution of actin filaments.
  • Y Yamane, H Shiga, H Asou, H Haga, K Kawabata, K Abe, E Ito
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 62 (4) 355 - 361 0914-9465 1999/10 [Not refereed][Not invited]
     
    We observed the time-dependent morphological alteration of astrocytes during their adhesion by atomic force microscopy (AFM) and investigated the relationships between this morphological alteration and the localization of actin filaments and connexin 43 by immunocytochemistry. The fine processes observed as fine ridge-like structures by AFM were closely concerned with ac tin filaments by immunocytochemistry, During the adhesion of astrocytes, actin filaments appeared to be aligned regularly beyond the borders among different cells. Detectable connexin immunoreactivity was changed in the following regions: 1) the tips of fine cell processes and the cell margin when astrocytes started to adhere; 2) the border of cells when astrocytes tightly adhered; and 3) non-specific sites when astrocytes became a cluster. In the former two cases, the immunopositive spots for connexin were observed to colocalize with the tips of cell processes with actin filaments. These results strongly suggest that connexin associated with actin filaments at the tip of cell processes plays an important role in the early stage of the adhesion of astrocytes. These observations afford valuable clues for understanding the glial communication.
  • T Hosono, M Yamanaka, T Tojima, Y Yamane, H Sadamoto, D Hatakeyama, H Haga, K Kawabata, K Abe, E Ito
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS SHORT NOTES & REVIEW PAPERS 38 (6B) 3940 - 3945 0021-4922 1999/06 [Not refereed][Not invited]
     
    As the first step in the study df morphological changes in neurons associated with their functional changes, we applied atomic force microscopy (AFM) for the observation of fine three-dimensional morphological changes in rat cerebellar granule cells stimulated by an agonist of glutamate receptors, N-methyl-D-aspartate (NMDA). The AFM revealed that NMDA changed the cross-sections of cell bodies from a trapezoid-like form to a triangle-like form within a minute. The fine hill-like structures on the top surfaces of the cell bodies became wider during the same period. These results were suggested to be induced by the depolymerization; of filamentous actin triggered by the entry of Ca2+ via cation channels complexed with the activated NMDA receptors.
  • Y Yamane, D Hatakeyama, H Haga, K Abe, E Ito
    ZOOLOGICAL SCIENCE 16 (1) 1 - 7 0289-0003 1999/02 [Not refereed][Not invited]
     
    The incomplete morphological characterization of type 2 astrocytes is in pari responsible for the slow progress of studies on these cells. To examine and characterize type 2 astrocytes morphologically, three-dimensional fine structures of the surfaces of type 2 astrocytes cultured from rat cerebella were studied by a combination of atomic force microscopic and immunocytochemical techniques. Atomic force microscopy (AFM) revealed irregular ridge-like structures that form a meshwork distributed throughout the cell body surfaces and the thick processes. These ridges were found to be of two heights (31 nm and 82 nm). This finding indicates two possible configurations responsible for shaping the meshwork: (1) two structures of different thickness are beneath the cell membrane; and (2) two structures are located at two different depths from the cell membrane. On the other hand, immunocytochemical studies for tubulin and glial fibrillary acidic protein (GFAP) revealed that these cytoskeletal filaments are similarly distributed within the resolution power of a light microscope. However, no detectable structures were obtained by actin staining. The immunocytochemical findings suggest that the AFM-revealed ridges forming the irregular meshwork on the cell surfaces may reflect very fine bundles of tubulin and/or GFAP. Therefore, AFM study, with the help of immunocytochemical study, is a powerful tool for characterizing cell morphology. The results of the present study reveal the first morphological characterization of type 2 astrocytes.
  • Time dependent viscoelastic image of living nerve cells using AFM
    K Kawabata, H Ishizuka, T Nitta, H Haga, E Ito, K Abe, T Ushiki, T Sambongi
    BIOLOGICAL PHYSICS 487 235 - 239 0094-243X 1999 [Not refereed][Not invited]
     
    The topographic and viscoelastic images of the nerve cell (NG108-15) living in the culture medium were successfully obtained in the force modulation mode using atomic force microscopy(AFM), We found that there exists variation of elasticity in the cell, To compare the elastic results with F-actin network of cytoskeleton which is considered as a possible origin for cell stiffness, the fluorescence observation was made on the fu;ed cells just after the AFM measurements. The results show no clear correspondence between density of F-actin and stiffness of the cells.
  • Thermal and structural study close to a smecticA-smecticA critical point
    P Gorria, P Barois, HT Nguyen, G Sigaud, L Navailles, CW Garland, H Haga
    MOLECULAR CRYSTALS AND LIQUID CRYSTALS SCIENCE AND TECHNOLOGY SECTION A-MOLECULAR CRYSTALS AND LIQUID CRYSTALS 330 1419 - 1426 1058-725X 1999 [Not refereed][Not invited]
     
    Two previous works([1,2]) firstly evidenced the possible occurrence of gaps of miscibility in smectic A solutions of two non polymer mesogens and later demonstrated the connection of this phenomenon with the difference in laver spacing of the two pure components. In the present paper we provide a detailed thermal and structural analysis of the phase separation around the smecticA-smecticA consolute point of a selected system. It encompasses two high temperature resolution studies which give valuable information about the universality class to which this critical point is likely to be attached.
  • Spatial and temporal viscoelastic imaging of living fibroblasts by atomic force microscopy
    H Haga, S Sasaki, K Kawabata, E Ito, K Abe, T Sambongi
    BIOLOGICAL PHYSICS 487 229 - 234 0094-243X 1999 [Not refereed][Not invited]
     
    We have investigated viscoelastic properties of living mouse fibroblasts (NIH3T3) in a physiological condition using both force mapping and force modulation modes in atomic force microscopy. Spatial distribution of the elastic moduli has been measured using the force mapping mode. The nucleus area of the cell is about 10 times softer than the cytoplasm. Temporal changes in shape and viscoelasticity of developing lamellipodia have been captured using the force modulation mode. The dynamic changes were succeeded in capturing with time resolution of 10 minutes.
  • H Haga, S Sasaki, M Morimoto, K Kawabata, E Ito, K Abe, T Sambongi
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS SHORT NOTES & REVIEW PAPERS 37 (6B) 3860 - 3863 0021-4922 1998/06 [Not refereed][Not invited]
     
    Using the force modulation mode in atomic force microscopy (AFM), we have succeeded in imaging elastic properties of agar gels immersed in water. The elastic images of ap ar have been captured simultaneously with the topographic images. Stiffer grains of agar whose size is about 200 nm can be clearly seen in the elastic image of 3.0% agar, while they are not so visible in the case of 1.5% agar. These grains probably correspond to aggregation of agar which cannot be observed in the topographic images. We also measured force-versus-distance curves using AFM to confirm that the absolute values of elastic modulus (Young's modulus) of agar coincide with the bulk values measured using the conventional stress-strain method. The estimated values of the elastic moduli with the AFM were 40 and 90 kPa for 1.5% and 3.0% agar gels, respectively. These are in good agreement with the respective bulk values of 30 and 80 kPa obtained using the conventional method.
  • S Sasaki, M Morimoto, H Haga, K Kawabata, E Ito, T Ushiki, K Abe, T Sambongi
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 61 (1) 57 - 63 0914-9465 1998/03 [Not refereed][Not invited]
     
    Using the force modulation mode in atomic force microscopy, we measured elastic properties of living mouse fibroblasts (NIH3T3) in a culture medium, The topographic images of the cellular surface and the corresponding elastic images of the cellular surface were able to be captured simultaneously with high spatial resolution, The consecutive images were useful for examining time-dependent changes in the cellular surface, We observed that some cells continued to shrink and change their softness for 2 hours, Then the force modulation mode in atomic force microscopy shows potential use in analyzing time-dependent regional elastic properties of living cells with high spatial resolution.
  • HAGA H, GARLAND C W
    PHYSICAL REVIEW E 57 (1) 603 - 609 1063-651X 1998 [Not refereed][Not invited]
  • F Beaubois, T Claverie, JP Marcerou, JC Rouillon, HT Nguyen, CW Garland, H Haga
    PHYSICAL REVIEW E 56 (5) 5566 - 5574 1063-651X 1997/11 [Not refereed][Not invited]
     
    The birefringence Delta n, the specific heat C-p, and the layer compressional elastic modulus B are reported for two liquid crystals near the nematic (N) to smectic-A (SmA) phase transition. As predicted long ago by MacMillan and de Gennes [P. G. de Gennes and J. Prost, The Physics of Liquid Crystals (Clarendon, Oxford, 1993)] the coupling of the nematic orientational order parameter to the smectic-A layering order parameter can substantially alter the critical behavior near the N-SmA transition if the nematic range is small and the nematic order parameter susceptibility is large. In this paper, we present a direct experimental comparison of two compounds: 4-octyloxy-4'-cyanobiphenyl (8OCB) with a short nematic range and 4-octyloxybenzoyloxy-4'-cyanotolane (C(8)tolane) with a very large N range. The temperature variations of the apparent birefringence hn and the specific heat C, across the N-SmA phase transition show the definite influence of the proximity of the isotropic phase in the case of 8OCB while the C(8)tolane behaves as expected for the three-dimensional XY universality class. The elastic modulus B in the SmA phase, measured at several wave vectors by the second-sound resonance technique, was studied with high resolution as a function of temperature on approaching T-c(N-SmA). These elastic data confirm the B leveling off in both cases with an apparent breakdown of hydrodynamics in the case of the C(8)tolane compound.
  • H Haga, CW Garland
    LIQUID CRYSTALS 23 (5) 645 - 652 0267-8292 1997/11 [Not refereed][Not invited]
     
    Bulk heptyloxybenzylidene butylaniline (70.4) undergoes first order nematic (N)-isotropic (I), nematic-smectic A (SmA), and smectic C (SmC)-crystal G(CrG) transitions as well as a mean-field second order SmA-SmC transition. The dispersion of 70 Angstrom diameter hydrophobic silica aerosil particles in 70.4 leads to doubling and significant temperature shifts for all three first order transitions, as determined with a.c. calorimetry. The SmA-SmC heat capacity peak merely shifts in position while remaining a single sharp Landau feature with an amplitude that decreases as the aerosil density increases. The behaviour of three 7O.4 + hydrophobic aerosil samples is discussed and compared to that of one 70.4 hydrophilic aerosil and previously reported results for 7O.4 + aerogel and 4O.8 + aerosil samples.
  • H Haga, CW Garland
    PHYSICAL REVIEW E 56 (3) 3044 - 3052 1063-651X 1997/09 [Not refereed][Not invited]
     
    High-resolution ac calorimetric studies show that the dispersion of 70-Angstrom-diameter hydrophilic silica spheres (aerosils) has a substantial effect on several liquid-crystal transitions in butyloxybenzlidene octylaniline (40.8). The weakly first-order nematic (N)-isotropic (I). the second-order nematic (N)-smectic-A (SmA), and the strongly first-order smectic-A (SmA)-crystal-B(CrB) freezing transition all exhibit shifted transition temperatures and substantial changes in the shape of excess heat capacity peaks. Power-law fits show an evolution of the N-SmA. critical exponent cu from alpha=0.135 in bulk 40.8 toward alpha approximate to alpha(XY)=-0.007 in 40.8+aerosil suspensions with silica densities rho(s) approximate to 0.08 g cm(-3). For rho(s) greater than or equal to 0.11 g cm(-3), the N-SmA Delta C-p peaks are rounded in a manner qualitatively like those for 40.8 confined in a high-porosity aerogel, one of which was also studied.
  • H Haga, CW Garland
    LIQUID CRYSTALS 22 (3) 275 - 277 0267-8292 1997/03 [Not refereed][Not invited]
     
    A high resolution a.c. and relaxation calorimetric study has been carried out on heptyloxybenzylidene butylaniline (7O.4) in two silica aerogels with mass densities rho=0.08 and 0.17 g cm(-3). Bulk 7O.4 exhibits strongly first order N-I, N-S-A and S-C-CrG transitions as well as a mean-field second order S-A-S-C transition. The 7O.4/aerogel samples exhibit three first order transitions (N-I, N-S-A, S-A-crystal) that are appreciably shifted and broadened relative to bulk 7O.4. The S-A-S-C transition is not observed in either of the aerogel samples.
  • HAGA H, KUTNJAK Z, IANNACCHIONE G S, QIAN S, FINOTELLO D, GARLAND C W
    PHYSICAL REVIEW E 56 (2) 1808 - 1818 1063-651X 1997 [Not refereed][Not invited]
  • Evaluation of the specific heat anomaly of RS(1-x)-ARS(x)
    N Noda, H Nakano, H Haga, R Nozaki, Y Shiozaki
    JOURNAL OF THE KOREAN PHYSICAL SOCIETY 29 S513 - S516 0374-4884 1996/11 [Refereed][Not invited]
     
    Thermal properties of RS(1-x)-ARS(x) studied by an ac calorimetric technique are reported. In order to obtain better values of the transition entropies for the mixed crystals, two problems have remained: evaluation of absolute value of the specific heat and precise determination of background of the very small anomaly. A method to evaluate the absolute value using the ac-calorimeter has been developed and determination of the background has been improved. Finally refinement of the value of the transition entropy are given for these mixed crystals.
  • Natsuko Noda, Hisashi Haga, Hidehiko Nakano, Ryusuke Nozaki, Yoichi Shiozaki
    FERROELECTRICS 184 293 - 296 0015-0193 1996 [Refereed][Not invited]
     
    The specific heat of the (Rochelle salt)(1-x) - (ammonium Rochelle salt)(x) mixed crystal system has been measured in the region III (0.18 <=.r less than or similar to 0.90) with an ac calorimetric technique. A very small anomaly has been observed around the transition temperature. It has been found that the transition entropy, roughly estimated from the anomalous specific heat, depends on x.
  • H HAGA, R NOZAKI, Y SHIOZAKI, K EMA
    JOURNAL OF THE PHYSICAL SOCIETY OF JAPAN 64 (11) 4258 - 4264 0031-9015 1995/11 [Refereed][Not invited]
     
    Heat capacity of K2ZnCl4 and K2SeO4 has been measured using a high-resolution ac calorimeter. Critical behavior of the heat capacity anomalies associated with the normal-incommensurate (N-INC) phase transitions has been analyzed with the preasymptotic renormalization-group expression which includes correction terms up to the second order. It is revealed that the heat capacity anomalies of K2ZnCl4 and K2SeO4 are described well with the three dimensional (3D) XY model; the critical exponent, the critical amplitude ratio and the dimensionless ratio derived from the first-order correction terms agree well with the theoretically expected values of 3D XY model. These results are consistent with our previous experimental result for Rb2ZnCl4 and the Cowley and Bruce's prediction that the N-INC phase transition is classified into the 3D XY system.
  • Akira Onodera, Oto Watanabe, Haruyasu Yamashita, Hisashi Haga, Yoichi Shiozaki
    FERROELECTRICS 155 311 - 316 0015-0193 1994 [Refereed][Not invited]
     
    Dielectric behavior in Thiourea was measured around ferroelectric phase III and in incommensurate phase IV. Very slow measurements revealed that a single peak of dielectric anomaly around phase III splits into two peaks. Time evolution of the dielectric constant was observed in incommensurate phase IV. In spite of the prediction of the DDW theory, dielectric constants do not decay exponentially, but follow a stretched exponential exp{-(t/tau)(beta)} with beta=0.4 and tau=680 hours. Both intrinsic process of kinetic evolution of discommensurations and extrinsic impurity diffusion with many metastable states are important for relaxation in the incommensurate phase.

Books etc

  • 三次元培養における培養手法と周辺技術動向
    芳賀 永 (Joint work三次元培養法(ハイドロゲル法))
    情報機構 2019/04
  • Chromosome Nanoscience and Technology
    TAYLOR & FRANCIS GROUP 2007

MISC

  • 上皮細胞シートの折り返しによる管腔形成と細胞極性
    芳賀 永  医学のあゆみ  268-  (6)  507  -511  2019/02  [Not refereed][Not invited]
  • HAGA Hisashi  Seibutsu Butsuri  58-  (4)  196  -199  2018  [Not refereed][Not invited]
     
    <p>Collective movement and 3D morphogenesis are essential events in diverse physiological processes. We found that epithelial cells (MDCK cells) move collectively along one direction on a soft collagen gel, whereas the cells migrate randomly on a rigid collagen-coated-glass. Moreover, lumen formation occurred when an epithelial sheet on a collagen gel was overlaid with another collagen gel. We also found that MDCK cells formed 3D morphology on top of the Matrigel. The appearance of the morphologies was like a tulip hat. The cells tugged at the peripheral matrix and remodeled the gel surface. In this mini-review, these dynamical behaviors of the epithelial cells are discussed in terms of the cellular contractile force and viscoelasticity of the extracellular matrix.</p>
  • Activating transcription factor 5 promotes cancer cell invasion through transcriptional activation of integrins
    A. Nukuda, H. Endoh, S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  26-  2015  [Not refereed][Not invited]
  • Lung cancer cells after irradiation indicate viability and malignancy dependent on activating transcription factor 5.
    S. Ishihara, M. Yasuda, A. Ishizu, H. Haga  MOLECULAR BIOLOGY OF THE CELL  25-  2014/12  [Not refereed][Not invited]
  • Rac1-, PI3K-, and integrin-beta 1-dependent leader cells that are essential for the collective migration of MDCK cells
    N. Yamaguchi, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  24-  2013  [Not refereed][Not invited]
  • EGFR and Integrin alpha 2 beta 1 Dependent Invasiveness of Lung Adenocarcinoma Cells that Survived 10 Gy Ionizing Radiation.
    X. Li, S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, T. Nishioka, H. Haga  MOLECULAR BIOLOGY OF THE CELL  23-  2012  [Not refereed][Not invited]
  • 細胞骨格・細胞運動・細胞移動 ラミニンを含む軟らかいゲル基質上でみられる上皮細胞集団の3次元形態形成(Cytoskeleton/ Cell motility/ Cell migration 3D Morphogenesis of Epithelial Cells on a Soft Substrate Containing Laminin Induced by Cellular Contractile Force)
    今井 美沙子, 水谷 武臣, 川端 和重, 芳賀 永  日本細胞生物学会大会講演要旨集  63回-  118  -118  2011/05  [Not refereed][Not invited]
  • 3D Morphogenesis of Epithelial Cells on a Laminin-rich Soft Substrate Induced by Cellular Contractile Force
    M. Imai, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  22-  2011  [Not refereed][Not invited]
  • Substrate stiffness regulates NF-kB expression and activity in cancer cells
    S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  22-  2011  [Not refereed][Not invited]
  • Increased Invasiveness and Non-spheroid Morphology Change of Human Lung Adenocarcinoma Cells That Survived 10 Gy Irradiation
    T. Nishioka, S. Ishihara, Y. Miyai, T. Mizutani, K. Kawabata, H. Shirato, R. Yamazaki, M. Yasuda, H. Haga, K. Kawabata  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  75-  (3)  S540  -S540  2009  [Not refereed][Not invited]
  • 水谷武臣, 芳賀永, 加藤幸作, 川端和重  細胞  40-  (6)  250  -253  2008/06/20  [Not refereed][Not invited]
  • HAGA Hisashi, MIZUTANI Takeomi, KAWABATA Kazushige  Journal of the Surface Science Society of Japan  29-  (4)  235  -238  2008/04/10  [Not refereed][Not invited]
  • Novel function of transcription factor ATF5: Blockade of p53-dependent apoptosis induced by irradiation
    T. Nishioka, M. Yasuda, H. Haga, R. Yamazaki, K. Tsutsumi, H. Shirato  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  72-  (1)  S43  -S43  2008  [Not refereed][Not invited]
  • MIZUTANI Takeomi, HAGA Hisashi, KAWABATA Kazushige  Biophysics  45-  (2)  105  -108  2005/03/25  [Not refereed][Not invited]
  • Collective movement of epithelial cells on collagen gel substrate
    H Haga, C Irahara, R Kobayashi, T Nakagaki, K Kawabata  BIOPHYSICAL JOURNAL  88-  (1)  429A  -429A  2005/01  [Not refereed][Not invited]
  • 水谷 武臣, 芳賀 永, 川端 和重  物性研究  83-  (3)  333  -334  2004/12/20  [Not refereed][Not 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。
  • Domain like structure of human-chromosome observed by mechanical scanning probe microscopy
    K Kawabata, K Nomura, H Haga, O Hoshi, D Fukushi, T Ushiki  MOLECULAR BIOLOGY OF THE CELL  15-  329A  -329A  2004/11  [Not refereed][Not invited]
  • Tensional homeostasis of a single fibroblast regulated by phosphorylation of the myosin regulatory light chain
    M Takeomi, H Haga, M Takahashi, K Kawabata  MOLECULAR BIOLOGY OF THE CELL  15-  159A  -159A  2004/11  [Not refereed][Not invited]
  • Cellular migration driven by relaxation of cortical tension imaged with mechanical force microscopy
    M Nagayama, H Haga, M Takahashi, K Kawabata  MOLECULAR BIOLOGY OF THE CELL  13-  193A  -193A  2002/11  [Not refereed][Not invited]
  • Stiffness change of living fibroblasts depending on cell motion
    M Nagayama, H Haga, K Kawabata  MOLECULAR BIOLOGY OF THE CELL  12-  171A  -171A  2001/11  [Not refereed][Not invited]

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2017 -2019 
    Author : Hisashi HAGA
  • Ministry of Education, Culture, Sports, Science and Technology:科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2015 -2019 
    Author : Hisashi HAGA
  • Ministry of Education, Culture, Sports, Science and Technology:科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2015 -2019 
    Author : Shigeru KONDO
  • Ministry of Education, Culture, Sports, Science and Technology:科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2015 -2019 
    Author : Shigeru KONDO
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2014 -2016 
    Author : Hisashi HAGA
  • 文部科学省:科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2014 -2015 
    Author : Hisashi HAGA
  • 文部科学省:科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2013 -2014 
    Author : Hisashi HAGA
  • 文部科学省:科学研究費補助金(基盤研究(A))
    Date (from‐to) : 2009 -2012 
    Author : Hiroki SHIRATO, 本間 さと, 玉木 長良, 芳賀 永, 但野 茂, 石川 正純
     
    放射線を軸に、ナノレベルの物理現象から社会的存在としての患者まで生体の動体追跡科学の基盤研究を行った。電子線トラック特性解析から細胞生存率モデルを提案した。DNAの二本鎖切断のうち修復されずに残るものの数がPoisson分布し、それが潜在的致死(PL)損傷であると仮定するNLP(Non-Lethal Probability)モデルによって、照射後の細胞の生存率をより適切に説明できた。細胞への放射線効果の予測を統計学的に行う際には、解析対象を分布値で表現する解析法を導入することで、より正しく記述できることが示唆された。ひと由来がん細胞が軟らかい基盤上の動きを観察したところNF-κBやLOXとの関係がわかり、細胞のゲル器質内への侵潤の3次元的観察系を構築する基礎が整った。マウス体内の各部位における遺伝子発現をリアルタイムに長期間、自由行動中の動物から計測する小動物内の分子の動体追跡技術を開発した。ルシフェラーゼレポーターを用い、体表からの微弱発光レベルの三次元空間における長期間追跡を行うことができ、定量的追跡への土台が整った。サルの脳定位照射・動体追跡照射の実験を可能にするために、準備的検討を行った。大動物の動体追跡実験が可能になると、生理学的検討に使える可能性が高い。ひとがん組織の放射線治療後の腫瘍の変化を定量的に記載し、予測するためのモデルを構築した。ひとがん腫瘤の動体追跡の...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2009 -2011 
    Author : Hisashi HAGA, 川端 和重
     
    細胞集団に自発的な協調運動を誘引させるため,コラーゲンゲルを作成し,その上に上皮細胞(MDCK細胞)を培養した.平成22年度では,細胞外基質との接着構造を解析し,リーダー細胞の出現機構について調べた.さらに,細胞間に「力」を伝達するセンサーの探索を行った.(1)細胞-基質間の接着構造の解析これまでの実験結果によって,軟らかいゲル基質上においては,リーダー細胞のみが基質との接着部位にintegrin-β1を発現し,リーダー細胞以外の細胞は別の接着タンパク質を発現していることが示唆されていた.平成22年度では,RT-PCRにより,リーダー細胞以外の細胞における接着構造の解明を目指した.しかし,integrin-β3,β4,β7など幾つか候補が見つかったものの,接着タンパク質の同定には至らなかったので,次年度も継続して実験を行う予定である.(2)リーダー細胞の出現機構の解明これまでの実験結果によって,基質の硬さに伴って上皮-間質転換(EMT)が誘引され,リーダー細胞が出現することが示唆されていた.平成22年度では,MDCK細胞のサブクローニングを行い,発現遺伝子と表現型を調べることでEMTを検証した.その結果,ガラス基盤上でも集団運動を示すサブクローンの獲得に成功した.そのサブクローンは細胞間接着が極めて強固であり,E-cadherinが有意に発現していることが明らかとなった.さら...
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    Date (from‐to) : 2009 -2010 
    Author : Isao TANAKA, 芳賀 永
     
    本研究課題では,タンパク質結晶化初期スクリーニングにおいて析出する極微小結晶様物質の力学的応答特性を,走査型プローブ顕微鏡を用いて測定するための研究開発を行った.前年度までに,走査型プローブ顕微鏡でタンパク質結晶を観察する系を構築し,さらに塩の結晶についても同様の実験系を考案した.本年度は,タンパク質結晶および塩結晶の測定数を増やすことに取り組んだ.タンパク質結晶については,ニワトリ卵白リゾチームと甘味タンパク質ソーマチンについては成功したが,確立した実験系ではかなり高濃度のタンパク質溶液が多量に必要となり,かつ通常の結晶化とは方式が異なるため,既存の結晶化戦略との溝を埋めるような研究が必要とされる.また本課題内で,結晶面の違いによって,力学的性質の差異が示唆されたため,異なる結晶面の特性を比較検討する研究が必要であることが見出された.塩類の結晶について本年度は,前年に成功したNaClと同様の方法によってさまざまな塩類を結晶化させることを試みたが,NaCl以外では測定に適した結晶を得ることはできず,タンパク質と塩類を詳細に比較するためには,異なる方法で結晶を得る必要がある.タンパク質とNaClの結晶の力学的応答を比較した場合,前者のほうが弾性に富むこと,またNaClの結晶は,表面に強い電荷を帯びていることが示唆され,結晶の判別に応用できることが期待された.しかし,タンパク質と...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2008 -2010 
    Author : Takeshi NISHIOKA, 芳賀 永, 安田 元昭, 本間 明宏, 堤香 織, 酒井 正春
     
    Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation". To better clarify the mechanism, gene expression profiling and pathological experiments were performed. A mouse fibrosarcoma cell line, QRsP, was used. Cells were irradiated with 10 Gy. Colony assay and cloning were performed. Six clones were established. cDNA analysis was performed on the clone that showed the largest number of colonies on the 2nd 10 Gy irradiation. Mouse transplantation experiment was then carried out. cDNA analysi...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2006 -2008 
    Author : Kazushige KAWABATA, 芳賀 永, 根本 幸児
     
    細胞集団がどのように連携・協同して血管や臓器などのマクロな形態を形成するかその機構を解明することを目的に、細胞に蓄積される力学的記憶効果を調べた。本研究では、走査型プローブ顕微鏡を用いた新たな測定装置を構築し、細胞に力学的変形刺激を加えた場合に細胞内に発生する力の応答を調べた。その結果、細胞の変形パターンによって、その後の細胞の力学的応答が大きく異なり、細胞レベルに力学的な記憶効果があることを明らかにした。
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2006 -2007 
    Author : Takeshi NISHIOKA, 安田 元昭, 芳賀 永, 本間 明宏, 堤 香織
     
    Novel function of transcription factor ATF5: blockade of p53-dependent apoptosis induced by irradiation.Purpose: p53-dependent cell death is considered as a predominant mechanism of tumor cell apoptosis induced by ionizing irradiation, and a large number of studies have shown that mutant p53-harboring tumor cells with a p53 gene mutation exhibit radioresistance. However, even tumor cells that express wild-type p53 display various degrees of radiosensitivity to ionizing irradiation. This indicates that there are additional pathways that affect p53-dependent cell death mechanisms. Here we des...
  • 文部科学省:科学研究費補助金(萌芽研究)
    Date (from‐to) : 2003 -2004 
    Author : Kazushige KAWABATA, 芳賀 永
     
    細胞運動や組織形成などには、基質の伸展刺激や流れずり応力等の力学的刺激が重要な働きをすることが知られている。本研究の目的は、外力に対する細胞の力学的応答を調べることにより、細胞内のアクチンフィラメントネットワークの動的性質を明らかにする、特に、外的刺激に対する力学的な平衡状態(張力ホメオスタシス)の存在およびその性質また分子レベルでの要因を明らかにすることである。本研究により得た結果は以下である。1)弾性シャーレを用いて、生きたマウス繊維芽細胞(NIH-3T3)を伸長もしくは収縮させた時の、細胞のかたさ分布の時間変化を力学SPMを用いて測定した。細胞内のアクチンストレスファイバーに働く張力にも恒常性が存在し、元の状態に戻るために2時間程度の時定数を持つ。2)ストレスファイバーの収縮力の起源であるミオシン調節軽鎖(MRLC ; Myosin Regulatory Light Chain)に結合したリン酸数と張力の応答の関係を調べた。1リン酸化はできるが2リン酸化することが出来ないMRLC(MRLC-T18A)を過剰に発現させた細胞を機械的に伸張させ、その力学的応答をSPMによって測定した。この細胞では細胞内張力ホメオスタシスは起こらない。3)wild-type細胞を伸長もしくは収縮し、リン酸化されたMRLCの細胞内の空間分布を共焦点蛍光顕微鏡で観察した。細胞の伸長直後にストレス...
  • 文部科学省:科学研究費補助金(若手研究(B))
    Date (from‐to) : 2002 -2004 
    Author : Hisashi HAGA
     
    前年度に開発した広域走査が可能な新型の走査型プローブ顕微鏡(WR-SPM)を用いて、上皮細胞が増殖し上皮組織を形成するまでの過程を力学的な見地から観察を行った。これまで市販のSPMでは100ミクロン程度の走査範囲が限界であったが、新たに開発したWR-SPMでは400ミクロンのエリアを走査することが可能となっている。現在までに、数百の細胞からなる上皮細胞のコロニー全体に対する弾性率分布の経時変化測定に成功している。さらに、一細胞レベルにおける細胞内張力のダイナミクスについてのSPM観察を行った。その結果、ストレスファイバーを構成するII型ミオシン調節軽鎖(MRLC)のリン酸化が細胞内張力の発生起源であり、伸長もしくは収縮といった外力が細胞に加わっても、細胞はMRLCのリン酸化レベルを変化させることで、細胞の形態や細胞内張力を安定化させていることが明らかとなった。また、保温装置付きの位相差顕微鏡を用いて、コラーゲンゲル基盤上で培養した上皮細胞が上皮コロニーを形成しながら集団で運動する過程を長時間観察した。その結果、(1)コラーゲン線維の配向性が上皮細胞の集団運動の方向を決定する。(2)ゲル基盤上の上皮細胞には、amoeboid型運動をするfollower細胞と、mesenchymal型運動をするleader細胞が存在し、それらの細胞の役割分担が空間的に制御されることで、上皮細胞は集団で一定方向に効率良く運動し、結合組織の形成を行う。ことなどが明らかとなった。以上、SPMで得られた結果と位相差顕微鏡による長時間観察の結果を比較すると、上皮細胞は組織を形成する過程において上皮コロニー周辺部のleader細胞が牽引力を発生し、その力がコロニー中心部に伝わることでコロニー全体が協調して一定方向に運動することが明らかとなった。
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2002 -2003 
    Author : Yoshihiko HIRAI, 芳賀 永, 川端 和重, 田中 芳雄, 川田 博昭, 菊田 久雄
     
    Understanding cellular migration as an integrated mechanical system requires an experimental investigations on mechanical properties of living cells. Force modulation mode with SPM is proposed as a useful method fur measuring stiffness of living cells with high temporal and spatial resolution. However, in liquid environment, cellular stiffness obtained by force modulation mode is often incredible.To clear this problem, numerical analysis of the 1-dimensional dynamic equation of a-micro cantilever, which concern about the visco-elastic property of the sample and viscosity of the liquid, is c...
  • 生細胞の組織形成過程における力学的制御機構の解明
    Date (from‐to) : 1996
  • Cell Mechanics and Regulatory Mechanism of Morphogenesis
    Date (from‐to) : 1996

Educational Activities

Teaching Experience

  • Soft Matter Medical Engineering
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : メカノバイオロジー、細胞生物学、生物物理学、医工学
  • Functional Cellular Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : メカノバイオロジー、細胞生物学、生物物理学、医工学
  • Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : ブレインストーミング、KJ法、アンチプロブレム、SWOT分析、プレゼンテーション
  • Idea Generation Workshop for Small Groups
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 生命科学院
    キーワード : ブレインストーミング、KJ法、アンチプロブレム、SWOT分析、プレゼンテーション
  • Statistical Mechanics for Life Science
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 統計力学
  • Quantum Mechanics for Life Science
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 量子力学、原子の構造


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