研究者情報

マスター

アカウント（マスター）

• 氏名

  芳賀　永(ハガ　ヒサシ), ハガ　ヒサシ

所属（マスター）

• 先端生命科学研究院　先端融合科学研究部門　細胞ダイナミクス科学分野

所属（マスター）

• 先端生命科学研究院　先端融合科学研究部門　細胞ダイナミクス科学分野

独自項目

syllabus

• 2021, ソフトマター科学実習, Laboratory Exercises in Soft Matter Science, 修士課程, 生命科学院, ソフトマター科学の研究技法
• 2021, ソフトマター科学研究, Research in Soft Matter Science, 修士課程, 生命科学院, ソフトマター科学研究、修士論文
• 2021, ソフトマター科学論文講読Ⅰ, Seminar in Soft Matter Science I, 修士課程, 生命科学院, セミナー、雑誌会、研究論文、リーディング
• 2021, ソフトマター科学論文講読Ⅱ, Seminar in Soft Matter Science II, 修士課程, 生命科学院, セミナー、雑誌会、研究論文、ライティング
• 2021, 生命医薬科学/生命融合科学/生命システム科学/ソフトマター科学 概論, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, ソフトマター医工学特論, Soft Matter Medical Engineering, 修士課程, 生命科学院, メカノバイオロジー、細胞生物学、生物物理学、医工学
• 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, ブレインストーミング、KJ法、プレゼンテーション、企業での研究開発
• 2021, 細胞機能科学特論, Functional Cellular Sciences, 修士課程, 生命科学院, メカノバイオロジー、細胞生物学、生物物理学、医工学
• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, 生命医薬科学概論, Biomedical and Pharmaceutical Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, ソフトマター科学概論, Soft Matter Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, 生命融合科学概論, Transdisciplinary Life Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, 生命システム科学概論, Biosystems Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, ソフトマター科学特別研究, Advanced Research in Soft Matter Science, 博士後期課程, 生命科学院, ソフトマター科学研究、博士論文
• 2021, ソフトマター科学論文講読, Seminar in Soft Matter Science, 博士後期課程, 生命科学院, セミナー、雑誌会、研究論文
• 2021, 博士海外研究, Doctral Research abroad, 博士後期課程, 生命科学院, 英語力、海外渡航、コミュニケーション、プレゼンテーション能力、異分野交流
• 2021, 国際共同研究提案演習, International Research Proposal Exercise, 博士後期課程, 生命科学院, ソフトマター科学研究、国際共同研究企画、提案書
• 2021, 国際学会口頭発表, International Research Presentation, 博士後期課程, 生命科学院, 国際学会、英語力、コミュニケーション、プレゼンテーション能力
• 2021, 生命科学文献講読, Literature Review for Life Science, 博士後期課程, 生命科学院, セミナー、雑誌会、研究論文
• 2021, 生命科学特別研究, Advanced Research in Life Science, 博士後期課程, 生命科学院, 生命科学研究、博士論文
• 2021, 研究提案演習, Research Proposal Exercise, 博士後期課程, 生命科学院, 英語力、海外渡航、コミュニケーション、プレゼンテーション能力、異分野交流
• 2021, 少人数討論型育成プログラム, Idea Generation Workshop for Small Groups, 博士後期課程, 生命科学院, ブレインストーミング、KJ法、プレゼンテーション、企業での研究開発
• 2021, 生物系の統計力学, Statistical Mechanics for Life Science, 学士課程, 理学部, 統計力学
• 2021, 生物系の量子力学, Quantum Mechanics for Life Science, 学士課程, 理学部, 量子力学、原子の構造

PositionHistory

• 大学院先端生命科学研究院長, 2023年4月1日, 2025年3月31日

researchmap

プロフィール情報

学位

• 博士（理学）（北海道大学）

プロフィール情報

• 姓

  芳賀, ハガ

• 名

  永, ヒサシ

• ID各種

  200901041795890455

対象リソース

https://altair.sci.hokudai.ac.jp/g3/member/haga_hisashi/

業績リスト

研究キーワード

• 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   Radiotherapy   粘弾性   カンチレバー   放射線   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   

研究分野

• ライフサイエンス / 腫瘍生物学
• ライフサイエンス / 形態、構造
• 自然科学一般 / 生物物理、化学物理、ソフトマターの物理
• ライフサイエンス / 細胞生物学
• ライフサイエンス / 生物物理学

経歴

• 2023年04月 - 現在 北海道大学 大学院先端生命科学研究院 研究院長
• 2013年05月 - 現在 北海道大学 大学院先端生命科学研究院 教授
• 2010年 - 2013年 - 北海道大学 大学院先端生命科学研究院 准教授
• 2007年 - 2010年 北海道大学 大学院理学院生命理学専攻 准教授
• 2006年 - 2007年 北海道大学 大学院理学院生命理学専攻 助教授
• 2002年 - 2006年 北海道大学 大学院理学研究科生物科学専攻 助教授
• 1997年 - 2002年 北海道大学 大学院理学研究科物理学専攻 助手
• 1995年 - 1997年 米国マサチューセッツ工科大学 化学科 博士研究員
• 1994年 - 1995年 日本学術振興会 特別研究員
• 1992年 - 1995年 北海道大学 教養部 非常勤講師

学歴

• 1992年04月 - 1995年03月   北海道大学   大学院理学研究科   物理学専攻 博士後期
• 1990年04月 - 1992年03月   北海道大学   大学院理学研究科   物理学専攻 修士課程
• 1986年10月 - 1989年03月   北海道大学   理学部   物理学科
• 1985年04月 - 1986年09月   北海道大学   理Ⅰ系

委員歴

• 2010年 - 現在   日本細胞生物学会   評議員   日本細胞生物学会
• 2020年04月 - 2022年03月   日本生物物理学会北海道支部   支部長
• 2016年 - 2020年   日本メカノバイオロジー学会   会長

論文

• Matrix stiffness regulates the triad communication of adipocytes/macrophages/endothelial cells through CXCL13.

  Arthur Choisez, Seiichiro Ishihara, Takuro Ishii, Yidan Xu, Sepideh D Firouzjah, Hisashi Haga, Ryoichi Nagatomi, Joji Kusuyama

  Journal of lipid research 100620 - 100620 2024年08月14日 [査読有り]
   

  Adipose tissue remodeling and plasticity are dynamically regulated by the coordinated functions of adipocytes, macrophages, and endothelial cells and extracellular matrix (ECM) that provides stiffness networks in adipose tissue component cells. Inflammation and fibrosis are crucial exogenous factors that dysregulate adipose tissue functions and drastically change the mechanical properties of the ECM. Therefore, communication among the ECM and adipose tissue component cells is necessary to understand the multifaceted functions of adipose tissues. To obtain in vivo stiffness, we utilized genipin as a crosslinker for collagen gels. Meanwhile, we isolated primary adipocytes, macrophages, and endothelial cells from C57BL/6J mice and incubated these cells in the differentiation media on temperature-responsive culture dishes. After the differentiation, these cell sheets were transferred onto genipin-crosslinked collagen gels with varying matrix stiffness. We found that inflammatory gene expressions were induced by hard matrix, whereas anti-inflammatory gene expressions were promoted by soft matrix in all three types of cells. Interestingly, the co-culture experiments of adipocytes, macrophages, and endothelial cells showed that the effects of soft or hard matrix stiffness stimulation on adipocytes were transmitted to the distant adipose tissue component cells, altering their gene expression profiles under normal matrix conditions. Finally, we identified that a hard matrix induces the secretion of CXCL13 from adipocytes, and CXCL13 is one of the important transmitters for stiffness communication with macrophages and endothelial cells. These findings provide insight into the mechano-transmission into distant cells and the application of stiffness control for chronic inflammation in adipose tissues with metabolic dysregulation.
• The EGF/EGFR axis and its downstream signaling pathways regulate the motility and proliferation of cultured oral keratinocytes.

  Ryota Kobayashi, Emi Hoshikawa, Taisuke Saito, Orakarn Suebsamarn, Eriko Naito, Ayako Suzuki, Seiichiro Ishihara, Hisashi Haga, Kei Tomihara, Kenji Izumi

  FEBS open bio 13 8 1469 - 1484 2023年05月27日 [査読有り]
   

  We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.
• Stiffness-Modulation of Collagen Gels by Genipin-Crosslinking for Cell Culture.

  Seiichiro Ishihara, Haruna Kurosawa, Hisashi Haga

  Gels (Basel, Switzerland) 9 2 2023年02月10日 [査読有り]
   

  The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.
• Substrate stiffness induces nuclear localization of myosin regulatory light chain to suppress apoptosis.

  Katsuya Onishi, Seiichiro Ishihara, Masayuki Takahashi, Akihiro Sakai, Atsushi Enomoto, Kentaro Suzuki, Hisashi Haga

  FEBS letters 2023年02月01日 [査読有り]
   

  Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P-MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P-MRLC. Zipper-interacting protein kinase [ZIPK; also known as death-associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P-MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P-MRLC via ZIPK. 2P-MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction.
• Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference.

  Sumire Ishida-Ishihara, Ryota Takada, Kazuya Furusawa, Seiichiro Ishihara, Hisashi Haga

  Scientific reports 12 1 20269 - 20269 2022年11月24日 [査読有り]
   

  Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.
• Mammaglobin 1 mediates progression of trastuzumab-resistant breast cancer cells through regulation of cyclins and NF-κB.

  Ratih Kusumastuti, Yuji Kumagai, Seiichiro Ishihara, Atsushi Enomoto, Takashi Murakami, Motoaki Yasuda, Hisashi Haga

  FEBS open bio 12 10 1797 - 1813 2022年07月29日 [査読有り]
   

  Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.
• Vasculature atrophy causes a stiffened microenvironment that augments epidermal stem cell differentiation in aged skin

  Ryo Ichijo, Koichiro Maki, Mio Kabata, Teruasa Murata, Arata Nagasaka, Seiichiro Ishihara, Hisashi Haga, Tetsuya Honda, Taiji Adachi, Takuya Yamamoto, Fumiko Toyoshima

  Nature Aging 2 7 592 - 600 2022年07月11日 [査読有り]
  
• The interferon-β/STAT1 axis drives the collective invasion of skin squamous cell carcinoma with sealed intercellular spaces

  Yuji Kumagai, Junko Nio-Kobayashi, Seiichiro Ishihara, Atsushi Enomoto, Masashi Akiyama, Ryosuke Ichihara, Hisashi Haga

  Oncogenesis 11 1 27 - 27 2022年05月24日 [査読有り]
   

  Abstract The process by which cancer cells invade as a cell cluster, known as collective invasion, is associated with metastasis and worse prognosis of cancer patients; therefore, inhibition of collective invasion is considered to improve cancer treatment. However, the cellular characteristics responsible for collective invasion remain largely unknown. Here, we successfully established subclones with various invasive potentials derived from human skin squamous carcinoma cells. The cell cluster of the highly invasive subclone had a hermetically sealed and narrow intercellular space. Interferon-β was localized to the sealed intercellular spaces, leading to collective invasion via the activation of signal transducer and activator of transcription 1 (STAT1). On the other hand, interferon-β was not localized to non-sealed and wide intercellular spaces of the cell cluster of low-invasive subclone with deficient STAT1 activity. In the mixed cell cluster of high- and low-invasive subclones, the high-invasive sub-clonal cells were located at the invasive front of the invasive protrusion, leading to collective invasion by the low-invasive sub-clonal cells. Tissue microarray analysis of human skin squamous cell carcinoma (SCC) also showed enrichment of STAT1 in the invasive front of SCCs. These findings indicate that the intercellular structure controls the potential for collective invasion via STAT1 regulation in SCC.
• Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics

  Tadashi Iida, Yasuyuki Mizutani, Nobutoshi Esaki, Suzanne M. Ponik, Brian M. Burkel, Liang Weng, Keiko Kuwata, Atsushi Masamune, Seiichiro Ishihara, Hisashi Haga, Kunio Kataoka, Shinji Mii, Yukihiro Shiraki, Takuya Ishikawa, Eizaburo Ohno, Hiroki Kawashima, Yoshiki Hirooka, Mitsuhiro Fujishiro, Masahide Takahashi, Atsushi Enomoto

  Oncogene 2022年04月13日 [査読有り]
  
• Matrix Stiffness Contributes to Cancer Progression by Regulating Transcription Factors.

  Seiichiro Ishihara, Hisashi Haga

  Cancers 14 4 2022年02月18日 [査読有り]
   

  Matrix stiffness is critical for the progression of various types of cancers. In solid cancers such as mammary and pancreatic cancers, tumors often contain abnormally stiff tissues, mainly caused by stiff extracellular matrices due to accumulation, contraction, and crosslinking. Stiff extracellular matrices trigger mechanotransduction, the conversion of mechanical cues such as stiffness of the matrix to biochemical signaling in the cells, and as a result determine the cellular phenotypes of cancer and stromal cells in tumors. Transcription factors are key molecules for these processes, as they respond to matrix stiffness and are crucial for cellular behaviors. The Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) is one of the most studied transcription factors that is regulated by matrix stiffness. The YAP/TAZ are activated by a stiff matrix and promotes malignant phenotypes in cancer and stromal cells, including cancer-associated fibroblasts. In addition, other transcription factors such as β-catenin and nuclear factor kappa B (NF-κB) also play key roles in mechanotransduction in cancer tissues. In this review, the mechanisms of stiffening cancer tissues are introduced, and the transcription factors regulated by matrix stiffness in cancer and stromal cells and their roles in cancer progression are shown.
• Soft surfaces promote astrocytic differentiation of mouse embryonic neural stem cells via dephosphorylation of MRLC in the absence of serum.

  Hiroshi Oyama, Akihiro Nukuda, Seiichiro Ishihara, Hisashi Haga

  Scientific reports 11 1 19574 - 19574 2021年10月01日 [査読有り]
   

  Astrocytes, which can be obtained from neural stem cells (NSCs) by adding serum and/or recombinant proteins in culture media or by passaging NSCs repeatedly, are expected to be applicable in regenerative medicine for the treatment of neurodegenerative diseases. However, astrocytes obtained using existing methods are costly and have poor quality. The stiffness of culture surfaces has been reported to affect astrocytic differentiation of adult NSCs. However, the influence of surface stiffness on astrocytic differentiation of embryonic NSCs has not yet been reported. In this study, we showed that astrocytic differentiation of embryonic NSCs was increased on soft surfaces (1 kPa and 12 kPa) compared with the NSCs on stiff surfaces (2.8 GPa) in serum-free condition. Furthermore, di-phosphorylated myosin regulatory light chain (PP-MRLC) was decreased in embryonic NSCs cultured on the soft surfaces than the cells on the stiff surfaces. Additionally, astrocytic differentiation of embryonic NSCs was induced by a Ras homolog associated kinase (ROCK) inhibitor, which decreased PP-MRLC in NSCs. These results suggest that decreasing the PP-MRLC of embryonic NSCs on soft surfaces or treating NSCs with a ROCK inhibitor is a good method to prepare astrocytes for application in regenerative medicine.
• Osmotic gradient induces stable dome morphogenesis on extracellular matrix.

  Sumire Ishida-Ishihara, Masakazu Akiyama, Kazuya Furusawa, Isao Naguro, Hiroki Ryuno, Takamichi Sushida, Seiichiro Ishihara, Hisashi Haga

  Journal of cell science 133 14 2020年06月23日 [査読有り][通常論文]
   

  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.
• Biophysical research in Hokkaido University, Japan.

  Tomoyasu Aizawa, Makoto Demura, Kazutoshi Gohara, Hisashi Haga, Koichiro Ishimori, Masataka Kinjo, Tamiki Komatsuzaki, Katsumi Maenaka, Min Yao

  Biophysical reviews 12 2 233 - 236 2020年04月 [査読有り][通常論文]
  
• Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis.

  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 2019年10月 [査読有り][通常論文]
   

  Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.
• Modulation and Characterization of the Double Network Hydrogel Surface-Bulk Transition

  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 2019年09月10日 [査読有り][通常論文]
  
• The intercellular expression of type-XVII collagen, laminin-332, and integrin-β1 promote contact following during the collective invasion of a cancer cell population.

  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 2019年07月 [査読有り][通常論文]
   

  Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-β1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-β1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-β1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-β1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.
• Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts.

  Yamamoto K, Otomo K, Nemoto T, Ishihara S, Haga H, Nagasaki A, Murakami Y, Takahashi M

  Experimental cell research 376 1 67 - 76 2019年03月 [査読有り][通常論文]
   

  Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.
• Mesenchymal actomyosin contractility is required for androgen-driven urethral masculinization in mice.

  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 - 95 2019年 [査読有り][通常論文]
   

  The morphogenesis of mammalian embryonic external genitalia (eExG) shows dynamic differences between males and females. In genotypic males, eExG are masculinized in response to androgen signaling. Disruption of this process can give rise to multiple male reproductive organ defects. Currently, mechanisms of androgen-driven sexually dimorphic organogenesis are still unclear. We show here that mesenchymal-derived actomyosin contractility, by MYH10, is essential for the masculinization of mouse eExG. MYH10 is expressed prominently in the bilateral mesenchyme of male eExG. Androgen induces MYH10 protein expression and actomyosin contractility in the bilateral mesenchyme. Inhibition of actomyosin contractility through blebbistatin treatment and mesenchymal genetic deletion induced defective urethral masculinization with reduced mesenchymal condensation. We also suggest that actomyosin contractility regulates androgen-dependent mesenchymal directional cell migration to form the condensation in the bilateral mesenchyme leading to changes in urethral plate shape to accomplish urethral masculinization. Thus, mesenchymal-derived actomyosin contractility is indispensable for androgen-driven urethral masculinization.
• Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization.

  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 2018年11月 [査読有り][通常論文]
   

  Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. Here, we studied the role of the actin-binding protein Girdin, a specific regulator of collective migration of neuroblasts in the brain, in collective cancer cell migration. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Finally, we showed the correlation of the expression of Girdin with that of the components of the E-cadherin complex and the differentiation of human skin cancer. Collectively, our results suggest that Girdin is an important modulator of the collective behavior of cancer cells.
• Anisotropic Multi-channel Collagen Gel (MCCG) Guides the Growth Direction of the Neurite-like Processes of PC12 Cells.

  Koh I, Furusawa K, Haga H

  Scientific reports 8 1 13901  2018年09月 [査読有り][通常論文]
  
• Long-term culture of human iPS cell-derived telencephalic neuron aggregates on collagen gel

  Hiroshi Oyama, Koji Takahashi, Yoshikazu Tanaka, Hiroshi Takemoto, Hisashi Haga

  Cell Structure and Function 43 1 85 - 94 2018年 [査読有り][通常論文]
   

  It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.
• Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

  Ishihara S, Aoki K, Mizutani T, Amano M, Nishimura SI, Haga H

  Cell structure and function 43 2 177 - 185 2018年 [査読有り][通常論文]
  
• Mathematical model of collective cell migrations based on cell polarity

  Masakazu Akiyama, Takamichi Sushida, Sumire Ishida, Hisashi Haga

  Development Growth and Differentiation 59 5 471 - 490 2017年06月01日 [査読有り][通常論文]
   

  Individual cells migrate toward the direction of the cell polarity generated by interior or exterior factors. Under situations without guides such as chemoattractants, they migrate randomly. On the other hand, it has been observed that cell groups lead to systematic collective cell migrations. For example, Dictyostelium discoideum and Madin-Darby canine kidney (epithelial) cells exhibit typical collective cell migration patterns such as uniformly directional migration and rotational migration. In particular, it has been suggested from experimental investigations that rotational migrations are intimately related to morphogenesis of organs and tissues in several species. Thus, it is conjectured that collective cell migrations are controlled by universal mechanisms of cells. In this paper, we review actual experimental data related to collective cell migrations on dishes and show that our self-propelled particle model based on the cell polarity can accurately represent actual migration behaviors. Furthermore, we show that collective cell migration modes observed in our model are robust.
• Utilization of the Student E-Portfolio in the Graduate Education: The Case Report of "Nitobe School Program" in Hokkaido University.

  Shotaro Imai, Michiyo Shimamura, Ankit A. Ravankar, Hisashi Haga, Yukinori Kobayashi, Yasuhiro Yamanaka

  6th IIAI International Congress on Advanced Applied Informatics(IIAI-AAI) 147 - 152 2017年
• Mesenchymal stem cells in breast cancer: Response to chemical and mechanical stimuli

  Seiichiro Ishihara, Suzanne M. Ponik, Hisashi Haga

  Oncoscience 4 11-12 158 - 159 2017年 [査読有り][通常論文]
  
• Conformational plasticity of JRAB/MICAL-L2 provides ‘law and order’ in collective cell migration

  Sakane A, Yoshizawa S, Nishimura M, Tsuchiya Y, Matsushita N, Miyake K, Horikawa K, Imoto I, Mizuguchi C, Saito H, Ueno T, Matsushita S, Haga H, Deguchi S, Mizuguchi K, Yokota H, Sasaki T

  Molecular Biology of the Cell 27 20 3095 - 3108 2016年10月 [査読有り][通常論文]
   

  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.
• Role of ATF5 in the invasive potential of diverse human cancer cell lines

  Akihiro Nukuda, Hiroki Endoh, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 474 3 509 - 514 2016年06月 [査読有り][通常論文]
   

  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.
• Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells

  Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata

  DATA IN BRIEF 6 793 - 798 2016年03月 [査読有り][通常論文]
   

  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.
• Heterogeneous filament network formation by myosin light chain isoforms effects on contractile energy output of single cardiomyocytes derived from human induced pluripotent stem cells

  Takeomi Mizutani, Kazuya Furusawa, Hisashi Haga, Kazushige Kawabata

  REGENERATIVE THERAPY 3 90 - 96 2016年03月 [査読有り][通常論文]
   

  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.
• Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers

  Yamada T, Tsuda M, Wagatsuma T, Fujioka Y, Fujioka M, Aya O. Satoh, Horiuchi K, Nishide S, Nanbo A, Totsuka Y, Haga H, Tanaka S, Shindoh M, Ohba Y

  Scientific reports 6 56878 1 - 16 2016年03月 [査読有り][通常論文]
   

  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.
• An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

  Seiichiro Ishihara, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  CYTOTECHNOLOGY 68 1 25 - 32 2016年01月 [査読有り][通常論文]
   

  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.
• Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

  Takeomi Mizutani, Rui Li, Hisashi Haga, Kazushige Kawabata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 465 2 270 - 274 2015年09月 [査読有り][通常論文]
   

  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.
• Stiff substrates increase YAP-signaling-mediated matrix metalloproteinase-7 expression

  A. Nukuda, C. Sasaki, S. Ishihara, T. Mizutani, K. Nakamura, T. Ayabe, K. Kawabata, H. Haga

  ONCOGENESIS 4 e165  2015年09月 [査読有り][通常論文]
   

  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.
• Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate

  Misako Imai, Kazuya Furusawa, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  SCIENTIFIC REPORTS 5 14208 1 - 10 2015年09月 [査読有り][通常論文]
   

  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.
• ATF5: development of oncogenic resistance to radiotherapy

  Seiichiro Ishihara, Hisashi Haga

  AGING-US 7 7 453 - 454 2015年07月 [査読有り][通常論文]
  
• 基質の硬さによって促進される大腸がん細胞の悪性化 転写因子YAPを介した基質分解酵素MMP-7の発現亢進

  温田 晃弘, 石原 誠一郎, 水谷 武臣, 中村 公則, 綾部 時芳, 川端 和重, 芳賀 永

  日本細胞生物学会大会講演要旨集 67回 168 - 168 (一社)日本細胞生物学会 2015年06月
• Compressive Stress Induces Dephosphorylation of the Myosin Regulatory Light Chain via RhoA Phosphorylation by the Adenylyl Cyclase/Protein Kinase A Signaling Pathway

  Kenji Takemoto, Seiichiro Ishihara, Takeomi Mizutai, Kazushige Kawabata, Hisashi Haga

  PLOS ONE 10 3 2015年03月 [査読有り][通常論文]
   

  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.
• Activating transcription factor 5 enhances radioresistance and malignancy in cancer cells

  Seiichiro Ishihara, Motoaki Yasuda, Akihiro Ishizu, Masayori Ishikawa, Hiroki Shirato, Hisashi Haga

  ONCOTARGET 6 7 4602 - 4614 2015年03月 [査読有り][通常論文]
   

  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.
• Secretion of different altered laminin isoforms results in three-dimensional morphological changes in cells cultured on Matrigel

  Misako Imai, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  2014 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2014 2015年01月09日 [査読有り][通常論文]
   

  Epithelial cells are known to form structures, such as cysts and tubes, with three-dimensional (3-D) morphologies. During formation of the morphologies of these structures, apico-basal polarity is regulated by secretion of laminins and integrins. In this study, we observed epithelial cells with mushroom-like 3-D morphologies many of these cells had tail structures, where endogenous laminin-α3, laminin-β1, and integrin-β1 were found to be accumulated. Interestingly, time-lapse observation of the cells showed rotational movement around the tail. We also observed the process of change from the single-cell morphology to the mushroom-like morphology. Additionally, we observed that single cells showed an elongated protrusion as a predecessor of the tail structure. In addition to laminin-α3, laminin-β1, and integrin-β1, laminin-β3 was also found to be localized in the protrusion. These results indicate that the apico-basal polarization and regulation of laminin secretion are crucial for the formation of the mushroom-like structure and for the rotational movement of Madin-Darby canine kidney (MDCK) cells cultured on Matrigel.
• Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and PI3K.

  Yamaguchi N, Mizutani T, Kawabata K, Haga H

  Scientific reports 5 7656  2015年01月 [査読有り][通常論文]
   

  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.
• Filamin B enhances the invasiveness of cancer cells into 3D collagen matrices

  Yuta Iguchi, Seiichiro Ishihara, Yoshimi Uchida, Kaori Tajima, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  Cell Structure and Function 40 2 61 - 67 2015年 [査読有り][通常論文]
   

  Numerous types of cancer cells migrate into extracellular tissues. This phenomenon is termed invasion, and is associated with poor prognosis in cancer patients. In this study, we demonstrated that filamin B (FLNb), an actin-binding protein, is highly expressed in cancer cell lines that exhibit high invasiveness, with a spindle morphology, into 3D collagen matrices. In addition, we determined that knockdown of FLNb in invasive cancer cells converts cell morphology from spindle-shaped, which is associated with high invasiveness, to round-shaped with low invasiveness. Furthermore, di-phosphorylation of myosin regulatory light chain (MRLC) and phosphorylation of focal adhesion kinase (FAK) are inhibited in FLNb-knockdown cancer cells. These results suggest that FLNb enhances invasion of cancer cells through phosphorylation of MRLC and FAK. Therefore, FLNb may be a new therapeutic target for invasive cancers.
• 大腸がん細胞において基質の硬さはミオシンのリン酸化を通じて基質分解酵素MMP7の発現を亢進する(Substrate stiffness enhances expression of matrix metalloproteinase 7 via myosin phosphorylation in colon cancer cells)

  温田 晃弘, 石原 誠一郎, 中村 公則, 綾部 時芳, 芳賀 永

  日本癌学会総会記事 73回 P - 3137 2014年09月
• Epithelial Sheet Folding Induces Lumen Formation by Madin-Darby Canine Kidney Cells in a Collagen Gel

  Sumire Ishida, Ryosuke Tanaka, Naoya Yamaguchi, Genki Ogata, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  PLOS ONE 9 8 e99655  2014年08月 [査読有り][通常論文]
   

  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.
• Modulation of extracellular conditions prevents the multilayering of the simple epithelium

  Takeomi Mizutani, Kazuki Takeda, Hisashi Haga, Mitsugu Todo, Kazushige Kawabata

  HISTOCHEMISTRY AND CELL BIOLOGY 141 5 473 - 481 2014年05月 [査読有り][通常論文]
   

  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.
• Modulation of extracellular conditions prevents the multilayering of the simple epithelium

  Takeomi Mizutani, Kazuki Takeda, Hisashi Haga, Mitsugu Todo, Kazushige Kawabata

  HISTOCHEMISTRY AND CELL BIOLOGY 141 5 473 - 481 2014年05月 [査読有り][通常論文]
   

  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.
• TRIM27/MRTF-B-dependent integrin β1 expression defines leading cells in cancer cell collectives.

  Kato T, Enomoto A, Watanabe T, Haga H, Ishida S, Kondo Y, Furukawa K, Urano T, Mii S, Weng L, Ishida-Takagishi M, Asai M, Asai N, Kaibuchi K, Murakumo Y, Takahashi M

  Cell reports 7 4 1156 - 1167 2014年05月 [査読有り][通常論文]
   

  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.
• Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture

  Hiro-taka Masuda, Seiichiro Ishihara, Ichiro Harada, Takeomi Mizutani, Masayori Ishikawa, Kazushige Kawabata, Hisashi Haga

  BIOTECHNIQUES 56 4 172 - 179 2014年04月 [査読有り][通常論文]
   

  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.
• 2P173 ミオシン結合タンパク質の消失は細胞が出す力に影響を与えるのか(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

  Li Rui, Mizutani Takeomi, Haga Hisashi, Kawabata Kazushige

  生物物理 54 1 S223  一般社団法人 日本生物物理学会 2014年
• Substrate stiffness regulates temporary NF-κB activation via actomyosin contractions.

  Ishihara S, Yasuda M, Harada I, Mizutani T, Kawabata K, Haga H

  Experimental cell research 319 19 2916 - 2927 2013年11月 [査読有り][通常論文]
   

  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.
• Lung cancer cells that survive ionizing radiation show increased integrin α2β1- and EGFR-dependent invasiveness.

  Li X, Ishihara S, Yasuda M, Nishioka T, Mizutani T, Ishikawa M, Kawabata K, Shirato H, Haga H

  PloS one 8 8 e70905  2013年08月 [査読有り][通常論文]
   

  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.
• Irradiation-tolerant lung cancer cells acquire invasive ability dependent on dephosphorylation of the myosin regulatory light chain

  Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga

  FEBS LETTERS 587 6 732 - 736 2013年03月 [査読有り][通常論文]
   

  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.
• Interruption of glycosphingolipid synthesis enhances osteoarthritis development in mice

  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 2012年08月 [査読有り][通常論文]
   

  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.
• 1PT230 Evaluation of microtubule deformation by live cell imaging and image analysis(The 50th Annual Meeting of the Biophysical Society of Japan)

  Tanaka Ryosuke, Mizutani Takeomi, Haga Hisashi, Kawabata Kazushige

  生物物理 52 S107  一般社団法人 日本生物物理学会 2012年
• Active fluctuation in the cortical cytoskeleton observed by high-speed live-cell scanning probe microscopy

  Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata

  ACTA BIOMATERIALIA 7 10 3766 - 3772 2011年10月 [査読有り][通常論文]
   

  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.
• Radiation-induced Cancer Cell Repopulation: A Possible Mechanism Implied by Experiments Using Transplantable Mouse-derived Sarcoma Cell Line

  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 2011年 [査読有り][通常論文]
   

  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.
• Nano-mechanical properties of living cells expressing constitutively active RhoA effectors

  Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 403 3-4 363 - 367 2010年12月 [査読有り][通常論文]
   

  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.
• Precise Synthesis and Physicochemical Properties of High-Density Polymer Brushes designed with Poly(N-isopropylacrylamide)

  Hiromasa Suzuki, Huda Muhammad Nurul, Takahiro Seki, Taisuke Kawamoto, Hisashi Haga, Kazushige Kawabata, Yukikazu Takeoka

  MACROMOLECULES 43 23 9945 - 9956 2010年12月 [査読無し][通常論文]
   

  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.
• Intermediate structure between chromatin fibers and chromosome revealed by mechanical stretching and SPM measurement

  Kensuke Ikeda, Takeomi Mizutani, Osamu Hoshi, Tatsuo Ushiki, Hisashi Haga, Kazushige Kawabata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 400 1 181 - 186 2010年09月 [査読無し][通常論文]
   

  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.
• Intermediate structure between chromatin fibers and chromosome revealed by mechanical stretching and SPM measurement

  Kensuke Ikeda, Takeomi Mizutani, Osamu Hoshi, Tatsuo Ushiki, Hisashi Haga, Kazushige Kawabata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 400 1 181 - 186 2010年09月 [査読有り][通常論文]
   

  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.
• Integrin beta 1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

  Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Takeshi Nishioka

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 396 3 651 - 655 2010年06月 [査読有り][通常論文]
   

  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.
• 1P130 染色体の牽引時に生じる力の振動の観察(核酸-構造・物性,第48回日本生物物理学会年会)

  HORII TAKUYA, IKEDA KENSUKE, MIZUTANI TAKEOMI, HAGA HISASHI, KAWABATA KAZUSHIGE

  生物物理 50 2 S42  一般社団法人 日本生物物理学会 2010年
• 2P218 デジタル画像相関法を用いた生きた細胞における細胞骨格の動態解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

  Mizutani Takeomi, Ishida Sumire, Hasemi Takatoshi, Hirakawa Yuuki, Kataoka Saya, Tanaka Ryosuke, Nishida Kazuki, Yahara Masao, Takeda Kazuki, Yoshimura Ryo, Kato Munetada, Doi Kenichi, Haga Hisashi, Kawabata Kazushige

  生物物理 50 2 S121  一般社団法人 日本生物物理学会 2010年
• Dynamics of leading lamellae of living fibroblasts visualized by high-speed scanning probe microscopy

  Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata

  HISTOCHEMISTRY AND CELL BIOLOGY 133 1 59 - 67 2010年01月 [査読有り][通常論文]
   

  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.
• Integrin β1-dependent Invasive Migration of Irradiation-tolerant Human Lung Adenocarcinoma Cells in 3D Collagen Matrix

  ISHIHARA Seiichiro, HAGA Hisashi, YASUDA Motoaki, MIZUTANI Takeomi, KAWABATA Kazushige, SHIRATO Hiroki, NISHIOKA Takeshi

  Biochemical and Biophysical Research Communications 396 3 651 - 655 2010年 [査読無し][通常論文]
   

  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 beta1 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 beta1 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 beta1-dependent phenotype, and integrin beta1 might be a potentially effective therapeutic target in combination with radiotherapy.
• Mechanical response of single myoblasts to various stretching patterns visualized by scanning probe microscopy

  Waka Mitsui, Kazushi Tamura, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata

  ARCHIVES OF HISTOLOGY AND CYTOLOGY 72 4-5 227 - 234 2009年12月 [査読有り][通常論文]
   

  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.
• Wide range scanning probe microscopy for probing mechanical effects on cellular function

  Takeomi Mizutani, Hisashi Haga, Kosaku Kato, Kazushige Kawabata

  ARCHIVES OF HISTOLOGY AND CYTOLOGY 72 4-5 235 - 243 2009年12月 [査読有り][通常論文]
   

  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.
• Regulation of Cellular Contractile Force in Response to Mechanical Stretch by Diphosphorylation of Myosin Regulatory Light Chain via RhoA Signaling Cascade

  Takeomi Mizutani, Kazushige Kawabata, Yoshikazu Koyama, Masayuki Takahashi, Hisashi Haga

  CELL MOTILITY AND THE CYTOSKELETON 66 7 389 - 397 2009年07月 [査読有り][通常論文]
   

  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 2009年02月 [査読有り][通常論文]
  
• Increased Motility and Invasiveness in Tumor Cells That Survive 10 Gy Irradiation

  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 2009年 [査読有り][通常論文]
   

  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.
• 3P-160 伸展刺激に対する細胞の硬化応答は細胞が2サイクル以上の周期伸展を記憶することで消失する(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

  Takemoto Kenji, Mitsui Waka, Tamura Kazushi, Mizutani Takeomi, Haga Hisashi, Kawabata Kazushige

  生物物理 49 S178  一般社団法人 日本生物物理学会 2009年
• 3P-155 印加する外力の周波数に依存した細胞の生理的応答(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

  Mizutani Takeomi, Onodera Shin, Haga Hisashi, Kawabata Kazushige

  生物物理 49 S177  一般社団法人 日本生物物理学会 2009年
• 3P-159 デジタル画像相開法により得られた局所伸展刺激下における細胞骨格ネットワークの不均一な変形分布(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

  Doi Kenichi, Mizutani Takeomi, Morita Yasuyuki, Uchino Masakazu, Todo Mitsugu, Haga Hisashi, Kawabata Kazushige

  生物物理 49 S178  一般社団法人 日本生物物理学会 2009年
• 3P-158 リーディングラメラにおける粒状機械構造のアクチン細胞骨格依存的なダイナミクス : 高速生細胞SPMを用いた研究(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

  Tamura Kazushi, Mizutani Takeomi, Haga Hisashi, Kawabata Kazushige

  生物物理 49 S177 - S178 一般社団法人 日本生物物理学会 2009年
• Displacement of actin filament and actin binding proteins under local deformation processed by digital image correlation method in a myoblast

  Takeomi Mizutani, Kenichi Doi, Yasuyuki Morita, Masakazu Uchino, Mitsugu Todo, Hisashi Haga, Kazushige Kawabata

  Journal of Biomechanical Science and Engineering 4 3 415 - 422 2009年 [査読有り][通常論文]
   

  Displacement of actin filament and its binding proteins in mouse myoblasts under locally applied deformation was analyzed by manual method or digital image correlation method. Cyotoskeletal components labeled by immunofluorescent technique or green fluorescent protein-fused protein were deformed via the movement of a glass needle which was poked into a cell. First, we confirmed the digital image correlation method is able to use to analyze displacement map by comparison of the manual method. Next, we examined whether the applied deformation isotropically propagates into cell body. At focal adhesions, fluorescent signals from the deformed area unchanged under the application. Mainly, focal adhesions around the poked area were moved to the direction of the movement of the needle. In addition, some adhesions away from the poked area were moved. Similar results were observed in phalloidin-stained cells. Finally, we applied the local deformation to live cells. However, displacement at the locally deformed area was not observed due to the disappearance of fluorescent signals. These results indicate that applied deformation propagated heterogeneously into a cell, and may imply that biochemical signals disrupt actin fibers under local deformation.
• Novel Function of Transcription Factor ATF5: Blockade of p53-dependent Apoptosis Induced by Ionizing Irradiation

  Takeshi Nishioka, Yusuke Miyai, Hisashi Haga, Kazushige Kawabata, Hiroki Shirato, Akihiro Homma, Kenichiro Shibata, Motoaki Yasuda

  CELL STRUCTURE AND FUNCTION 34 1 17 - 22 2009年 [査読有り][通常論文]
   

  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.
• Mechanical response to isotropic shrinkage of fibroblasts measured by scanning probe microscopy

  Taisuke Kawamoto, Hisashi Haga, Kazushi Tamura, Takeomi Mizutani, Kazushige Kawabata

  JAPANESE JOURNAL OF APPLIED PHYSICS 47 7 6173 - 6176 2008年07月 [査読無し][通常論文]
   

  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.
• 1S8-2 細胞の集団運動と組織化のダイナミクス(1S8 細胞協同運動における細胞内および細胞間の力学的コミュニケーション,第46回日本生物物理学会年会)

  芳賀 永

  生物物理 48 S6 - S7 一般社団法人 日本生物物理学会 2008年
• 3P-167 ストレスファイバー形成過程において出現するアクチン線維凝集体 : アクチン線維ネットワーク再編成に対してアクチン線維凝集体が果たし得る役割(細胞生物学的課題(3),第46回日本生物物理学会年会)

  Tamura Kazushi, Mizutani Takeomi, Haga Hisashi, Kawabata Kazushige

  生物物理 48 S153  一般社団法人 日本生物物理学会 2008年
• 2P-250 走査型プローブ顕微鏡による生きた海馬神経細胞シナプスの3次元形態観察(神経・感覚,第46回日本生物物理学会年会)

  JIANG Jun, MIZUTANI Takeomi, HAGA Hisashi, KUDOH Suguru, TAGUCHI Takahisa, KAWABATA Kazushige

  生物物理 48 S113  一般社団法人 日本生物物理学会 2008年
• Matrixmetalloproteinases: Up-regulated in subclones that survived 10-Gy irradiation

  Takeshi Nishioka, Motoaki Yasuda, Kaori Tsutsumi, Hisashi Haga, Hiroki Shirato

  Radiation Medicine - Medical Imaging and Radiation Oncology 25 8 430 - 431 2007年10月 [査読有り][通常論文]
  
• Visualization of stretch-induced intracellular tensional response of single fibroblasts by mechanical scanning probe microscopy

  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 2007年08月 [査読無し][通常論文]
   

  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.
• Development of a device to stretch tissue-like materials and to measure their mechanical properties by scanning probe microscopy

  Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata

  ACTA BIOMATERIALIA 3 4 485 - 493 2007年07月 [査読有り][通常論文]
   

  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.
• Imaging mechanical properties of living cells by scanning probe microscopy

  Hisashi Haga, Masafumi Nagayama, Kazushige Kawabata

  CURRENT NANOSCIENCE 3 1 97 - 103 2007年02月 [査読無し][通常論文]
   

  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.
• 3P192 上皮細胞の協調的集団運動を誘引するコラーゲン基質上での細胞形態および細胞-基質間接着(細胞生物学的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

  小椋 隼人, 林 雅名子, 水谷 武臣, 川端 和重, 芳賀 永

  生物物理 47 S251  一般社団法人 日本生物物理学会 2007年
• 3P199 アクチンストレスファイバーに沿って運動する細胞内顆粒 : 走査型プローブ顕微鏡によるタイムラプス観察(細胞生物学的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

  田村 和志, 水谷 武臣, 芳賀 永, 川端 和重

  生物物理 47 S252  一般社団法人 日本生物物理学会 2007年
• 1P130 力学的牽引とSPMを用いたクロマチン線維-染色体間に存在する中間構造の発見(蛋白質(蛋白質工学・進化工学)、核酸結合蛋白質、核酸,口頭発表,第45回日本生物物理学会年会)

  池田 健佑, 星 治, 牛木 辰男, 水谷 武臣, 芳賀 永, 川端 和重

  生物物理 47 S56  一般社団法人 日本生物物理学会 2007年
• 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 2007年01月 [査読有り][通常論文]
  
• 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 2007年 [査読無し][通常論文]
  
• Collective Movement and Morphogenesis of Epithelial Cells

  Proceedings of the international symposium on topological aspects of critical systems and networks 82 - 85 2007年 [査読無し][通常論文]
  
• Diphosphorylation of the myosin regulatory light chain enhances the tension acting on stress fibers in fibroblasts

  Takeomi Mizutani, Hisashi Haga, Yoshikazu Koyama, Masayuki Takahashi, Kazushige Kawabata

  JOURNAL OF CELLULAR PHYSIOLOGY 209 3 726 - 731 2006年12月 [査読有り][通常論文]
   

  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.
• The role of actin-binding protein filamin A in cellular stiffness and morphology studied by wide-range scanning probe microscopy

  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 2006年03月 [査読無し][通常論文]
   

  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.
• Observation of stiff domain structure on collagen gels by wide-range scanning probe microscopy

  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 2006年03月 [査読無し][通常論文]
   

  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.
• 2P529 Tensional Responses of Single Living Cells to External Stretch Visualized by SPM(52. Bio-imaging,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Tamura Kazushi, Mizutani Takeomi, Haga Hisashi, Kawabata Kazushige

  生物物理 46 2 S428  一般社団法人 日本生物物理学会 2006年
• 2P176 New filamentous ordered structure appeared by stretching of unfixed human chromosome(36. DNA to chromatin,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Ikeda Kensuke, Hoshi Osamu, Ushiki Tatsuo, Haga Hisashi, Kawabata Kazushige

  生物物理 46 2 S339  一般社団法人 日本生物物理学会 2006年
• Static friction of agar gels: Formation of contact junctions at frictional interface

  T Nitta, H Kato, H Haga, K Nemoto, K Kawabata

  JOURNAL OF THE PHYSICAL SOCIETY OF JAPAN 74 11 2875 - 2879 2005年11月 [査読無し][通常論文]
   

  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.
• Visualization of elasticity distribution of single human chromosomes by scanning probe microscopy

  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 2005年07月 [査読無し][通常論文]
   

  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.
• Spatiotemporal variation in cellular stiffness corresponding to development of morphological polarity

  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 2005年07月 [査読無し][通常論文]
   

  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.
• Collective movement of epithelial cells on a collagen gel substrate

  H Haga, C Irahara, R Kobayashi, T Nakagaki, K Kawabata

  BIOPHYSICAL JOURNAL 88 3 2250 - 2256 2005年03月 [査読有り][通常論文]
   

  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.
• 2SD05 張力ホメオスタシス : 伸縮刺激に対する細胞内張力の安定化機構(細胞レベルの機能解明に向けた力学的なアプローチ)

  水谷 武臣, 芳賀 永, 高橋 正行, 小山 芳一, 川端 和重

  生物物理 45 S22  一般社団法人 日本生物物理学会 2005年
• 3SF52 ナノフォース走査型プローブ顕微鏡の開発と細胞生物学への応用(バイオとナノの融合 : 生物物理学からのアプローチ)(北海道大学21世紀COE共催シンポジウム)

  芳賀 永

  生物物理 45 S30  一般社団法人 日本生物物理学会 2005年
• 2P129 力学的牽引における染色体のステップ的伸張(核酸 A) 構造・物性))

  池田 健佑, 星 治, 牛木 辰男, 芳賀 永, 川端 和重

  生物物理 45 S152  一般社団法人 日本生物物理学会 2005年
• 1P338 生細胞の伸縮変形に対する力学的応答 : 走査型プローブ顕微鏡を用いた測定系の開発(バイオイメージング))

  田村 和志, 水谷 武臣, 芳賀 永, 川端 和重

  生物物理 45 S116  一般社団法人 日本生物物理学会 2005年
• Cellular stiffness response to external deformation: Tensional homeostasis in a single fibroblast

  T Mizutani, H Haga, K Kawabata

  CELL MOTILITY AND THE CYTOSKELETON 59 4 242 - 248 2004年12月 [査読有り][通常論文]
   

  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.
• Contribution of cellular contractility to spatial and temporal variations in cellular stiffness

  M Nagayama, H Haga, M Takahashi, T Saitoh, K Kawabata

  EXPERIMENTAL CELL RESEARCH 300 2 396 - 405 2004年11月 [査読有り][通常論文]
   

  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.
• Wide-range scanning probe microscopy for visualizing biomaterials in the submillimeter range

  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 2004年07月 [査読無し][通常論文]
   

  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.
• 2P203 ミオシン調節軽鎖のリン酸化に基づいた細胞の張力ホメオスタシス機構(細胞生物的課題 : 接着・運動・骨格・伝達・膜)

  水谷 武臣, 芳賀 永, 高橋 正行, 川端 和重

  生物物理 44 S160  一般社団法人 日本生物物理学会 2004年
• 細胞のマイクロ粘弾性解析

  永山 昌史, 芳賀 永, 田中 芳雄, 平井 義彦, 川端 和重

  電子顕微鏡 38 2 90 - 93 社団法人 日本顕微鏡学会(The Japanese Society of Microscopy) 2003年07月31日
• 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 2003年03月 [査読無し][通常論文]
   

  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.
• 張力によって協調された細胞運動

  永山 昌史, 芳賀 永, 高橋 正行, 川端 和重

  生物物理 43 S108  一般社団法人 日本生物物理学会 2003年
• コラーゲンゲル線維の配向がもたらす上皮細胞の集団運動

  伊良原 史子, 芳賀 永, 川端 和重

  生物物理 43 S109  一般社団法人 日本生物物理学会 2003年
• 伸縮刺激に対する繊維芽細胞の張力回復現象

  水谷 武臣, 芳賀 永, 川端 和重

  生物物理 43 S162  一般社団法人 日本生物物理学会 2003年
• Microdomain structure of agar gels observed by mechanical-scanning probe microscopy

  T Nitta, Y Endo, H Haga, K Kawabata

  JOURNAL OF ELECTRON MICROSCOPY 52 3 277 - 281 2003年 [査読有り][通常論文]
   

  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.
• Time dependent static friction force of agar gel-on-glass plate immersed in water

  T Nitta, H Haga, K Kawabata

  JOURNAL DE PHYSIQUE IV 12 PR9 319 - 320 2002年11月 [査読無し][通常論文]
   

  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.
• Improvement of force modulation mode with scanning probe microscopy for imaging viscoelasticity of living cells

  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 2002年07月 [査読無し][通常論文]
   

  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.
• 3N0915 軟らかいコラーゲンゲル上で特異的にみられる上皮細胞の極性形成と集団運動(12.細胞生物学的課題,一般講演,日本生物物理学会第40回年会)

  芳賀 永, 平川 直, 伊良原 史子, 林 雅名子, 高橋 正行, 川端 和重

  生物物理 42 2 S194  一般社団法人 日本生物物理学会 2002年
• 3G0900 弾性シャーレを用いた外力印加に対する繊維芽細胞のかたさ応答(27.バイオイメージング,一般講演,日本生物物理学会第40回年会)

  水谷 武臣, 芳賀 永, 川端 和重

  生物物理 42 2 S162  一般社団法人 日本生物物理学会 2002年
• 3G0930 走査型プローブ顕微鍍による接着面積を制限した繊維芽細胞のかたさ測定(27.バイオイメージング,一般講演,日本生物物理学会第40回年会)

  宮下 博樹, 芳賀 永, 川端 和重

  生物物理 42 2 S163  一般社団法人 日本生物物理学会 2002年
• Drastic change of local stiffness distribution correlating to cell migration in living fibroblasts

  M Nagayama, H Haga, K Kawabata

  CELL MOTILITY AND THE CYTOSKELETON 50 4 173 - 179 2001年12月 [査読無し][通常論文]
   

  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.
• Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts

  T Saitoh, S Takemura, K Ueda, H Hosoya, M Nagayama, H Haga, K Kawabata, A Yamagishi, M Takahashi

  FEBS LETTERS 509 3 365 - 369 2001年12月 [査読無し][通常論文]
   

  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.
• 3P194細胞運動における力学的な協調機構

  永山 昌史, 芳賀 永, 川端 和重

  生物物理 41 S207  一般社団法人 日本生物物理学会 2001年
• 1P242SPMによる機械刺激に対する細胞の力学応答観察

  佐戸 康洋, 芳賀 永, 川端 和重

  生物物理 41 S93  一般社団法人 日本生物物理学会 2001年
• Evaluation of Viscoelasticity of Biological Cells in Liquid by Scanning Probe Microscopy

  Yoshio Tanaka, Yoshihiko Hirai, Masaaki Kabuto, Kazushige Kawabata, Hisashi Haga, Masahumi Nagayama

  Nihon Kikai Gakkai Ronbunshu, C Hen/Transactions of the Japan Society of Mechanical Engineers, Part C 67 663 3498 - 3504 2001年 [査読有り][通常論文]
   

  An analytical method is proposed to evaluate the viscoelasticity of a biomaterial in water by the scanning probe microscope. The proposed method utilizes the dependence of amplitude and phase lag of cantilever vibration on the viscoelasticity when the tip on the cantilever cyclically penetrates into the cell. The cyclical penetrations are carried out in two ways one is by excitation of the cantilever fixed end, and other by that of the table. The analysis takes the drag force of water due to the cantilever vibration into account. The stiffness and the viscosity of the cell are analytically related with the amplitude and the phase lag of the cantilever vibration. The results shows that the relations among the amplitude, the phase lag, stiffness and viscosity vary significantly depending on the excitation frequencies of the cantilever fixed end and the table. © 2001, The Japan Society of Mechanical Engineers. All rights reserved.
• Mechanical effects on collective phenomena of biological systems: cell locomotion

  Kazushige Kawabata, Masafumi Nagayama, Hisashi Haga, Takashi Sambongi

  CURRENT APPLIED PHYSICS 1 1 66 - 71 2001年01月 [査読無し][通常論文]
   

  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.
• 細胞運動に伴う力学構造のAFM 粘弾性測定

  芳賀 永, 川端 和重

  電子顕微鏡 35 3 276 - 278 The Japanese Society of Microscopy 2000年11月30日
• Mechanical properties of membrane surface of cultured astrocyte revealed by atomic force microscopy

  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 2000年06月 [査読無し][通常論文]
   

  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.
• Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton

  H Haga, S Sasaki, K Kawabata, E Ito, T Ushiki, T Sambongi

  ULTRAMICROSCOPY 82 1-4 253 - 258 2000年02月 [査読無し][通常論文]
   

  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.
• Comparing microscopic with macroscopic elastic properties of polymer gel

  T Nitta, H Haga, K Kawabata, K Abe, T Sambongi

  ULTRAMICROSCOPY 82 1-4 223 - 226 2000年02月 [査読無し][通常論文]
   

  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.
• 1J1715 生きた細胞におけるかたさ分布の時間発展

  永山 昌史, 芳賀 永, 川端 和重

  生物物理 40 S80  一般社団法人 日本生物物理学会 2000年
• 3F1145 ハイブリッドAFMによる生きた細胞の細胞骨格分布と弾性率分布の観測

  佐戸 康洋, 芳賀 永, 松岡 一郎, 川端 和重

  生物物理 40 S182  一般社団法人 日本生物物理学会 2000年
• Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy

  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 2000年 [査読無し][通常論文]
   

  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 2000年 [査読無し][通常論文]
   

  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 2000年 [査読無し][通常論文]
   

  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 2000年 [査読無し][通常論文]
   

  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.
• Dynamics of astrocyte adhesion as analyzed by a combination of atomic force microscopy and immunocytochemistry: the involvement of actin filaments and connexin 43 in the early stage of adhesion

  Y Yamane, H Shiga, H Asou, H Haga, K Kawabata, K Abe, E Ito

  ARCHIVES OF HISTOLOGY AND CYTOLOGY 62 4 355 - 361 1999年10月 [査読無し][通常論文]
   

  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.
• 原子間力顕微鏡による生きた細胞の粘弾性マッピング

  川端 和重, 芳賀 永, 三本木 孝

  日本バイオレオロジー学会誌(B&R) = Journal of Japanese Society of Biorheology 13 3 29 - 37 特定非営利活動法人 日本バイオレオロジー学会 1999年09月30日
• Atomic force microscopic observation of three-dimensional morphological changes of neurons where stimulated by a neurotransmitter

  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 1999年06月 [査読無し][通常論文]
   

  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.
• 原子間力顕微鏡をもちいた培養細胞の粘弾性測定

  川端 和重, 芳賀 永

  生物物理 39 3 179 - 181 一般社団法人日本生物物理学会 1999年05月
• Surface structures of cultured type 2 astrocytes revealed by atomic force microscopy

  Y Yamane, D Hatakeyama, H Haga, K Abe, E Ito

  ZOOLOGICAL SCIENCE 16 1 1 - 7 1999年02月 [査読無し][通常論文]
   

  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.
• 1PA095 グリア細胞の細胞間接着過程についてのAFMおよび免疫細胞化学的解析

  山根 ゆか子, 志賀 葉月, 阿相 皓晃, 芳賀 永, 川端 和重, 阿部 和厚, 伊藤 悦朗

  生物物理 39 S57  一般社団法人 日本生物物理学会 1999年
• 1PA013 生きた細胞のAFM弾性マッピングと細胞骨格の免疫蛍光観察

  芳賀 永, 佐々木 茂雄, 川端 和重, 伊藤 悦朗, 牛木 辰男, 三本木 孝

  生物物理 39 S37  一般社団法人 日本生物物理学会 1999年
• 1PA014 AFM Force Modulation法による生きた細胞の粘弾性測定

  永山 昌史, 芳賀 永, 川端 和重, 伊藤 悦朗, 三本木 孝

  生物物理 39 S37  一般社団法人 日本生物物理学会 1999年
• 1PA012 原子間力顕微鏡を用いた弾性測定 : 定量性の確認

  新田 高洋, 芳賀 永, 川端 和重, 阿部 和厚, 三本木 孝

  生物物理 39 S36  一般社団法人 日本生物物理学会 1999年
• 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 1999年 [査読無し][通常論文]
   

  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 1999年 [査読無し][通常論文]
   

  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 1999年 [査読無し][通常論文]
   

  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.
• Imaging elastic properties of soft materials immersed in water using force modulation mode in atomic force microscopy

  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 1998年06月 [査読無し][通常論文]
   

  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.
• Elastic properties of living fibroblasts as imaged using force modulation mode in atomic force microscopy

  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 1998年03月 [査読無し][通常論文]
   

  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.
• Calorimetric study of the smectic-A-hexatic-B transition in 3(10)OBC

  H Haga, CW Garland

  PHYSICAL REVIEW E 57 1 603 - 609 1998年01月 [査読有り][通常論文]
   

  High-resolution calorimetry has been used to study the smectic-A(Sm-A)-hexatic-B(Hex-B) transition in n-propyl-4'-n-decyloxybiphenyl-4-carboxylate [3(10)OBC]. This transition is clearly first order with substantial pretransitional heat capacity wings. Power law fits to Delta C-p are possible and yield an effective "critical" exponent alpha(eff)=0.68+/-0.10 and a discontinuity in the "critical" background term B-c. These results are compatible with those recently reported for the homolog 650BC, where the first order character is very weak and subtle. The nature of the Sm-A-Hex-B transition appears to be quasicritical, which could be the result of a coupling between the amplitude of the bond-orientational order and in-plane positional strain.
• Calorimetric study of the smectic-A－hexatic-B transition in 3(10)OBC

  HAGA H, GARLAND C W

  PHYSICAL REVIEW E 57 1 603 - 609 1998年 [査読無し][通常論文]
  
• Influence of nematic range on birefringence, heat capacity and elastic modulus near a nematic-smectic-A phase transition

  F Beaubois, T Claverie, JP Marcerou, JC Rouillon, HT Nguyen, CW Garland, H Haga

  PHYSICAL REVIEW E 56 5 5566 - 5574 1997年11月 [査読無し][通常論文]
   

  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.
• Thermal study of the influence of aerosils on the phase transitions of heptyloxybenzylidene butylaniline

  H Haga, CW Garland

  LIQUID CRYSTALS 23 5 645 - 652 1997年11月 [査読無し][通常論文]
   

  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.
• Effect of silica aerosil particles on liquid-crystal phase transitions

  H Haga, CW Garland

  PHYSICAL REVIEW E 56 3 3044 - 3052 1997年09月 [査読無し][通常論文]
   

  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.
• Calorimetric study of heptyloxybenzylidene butylaniline in silica aerogels

  H Haga, CW Garland

  LIQUID CRYSTALS 22 3 275 - 277 1997年03月 [査読無し][通常論文]
   

  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.
• Quasicritical heat capacity at a smectic-A-hexatic-B phase transition

  HAGA H, KUTNJAK Z, IANNACCHIONE G S, QIAN S, FINOTELLO D, GARLAND C W

  PHYSICAL REVIEW E 56 2 1808 - 1818 1997年 [査読無し][通常論文]
  
• 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 1996年11月 [査読有り][通常論文]
   

  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.
• Thermal properties of improper ferroelectric Tb2(MoO4)3: Critical phenomena and traces of isomorphic phase transition

  B. A. Strukov, A. Onodera, S. A. Taraskin, I. V. Shnaidshtein, B. S. Red'kin, H. Haga

  Ferroelectrics 185 1-4 181 - 184 1996年 [査読有り][通常論文]
   

  The specific heat of Tb2(MoO4)3 single crystal was measured in the temperature region 85-56OK by ac- and adiabatic calorimetry. Two anomalies were revealed after careful subtraction of the background heat capacity and were attributed to an improper ferroelectric phase transition (T0 = 429.79K) and to a possible overcritical trace of an isomorphic phase transition (270K). The critical exponents for T0 were determined as α' = 0.5 , α = 1.35 it is suggested that the latter exponent is connected with the "frozen in" charged point defects.
• A CALORIMETRIC STUDY OF THE (ROCHELLE SALT)(1-x) - (AMMONIUM ROCHELLE SALT)(x) MIXED CRYSTAL SYSTEM

  Natsuko Noda, Hisashi Haga, Hidehiko Nakano, Ryusuke Nozaki, Yoichi Shiozaki

  FERROELECTRICS 184 293 - 296 1996年 [査読有り][通常論文]
   

  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.
• PREASYMPTOTIC 3D XY CRITICAL-BEHAVIOR OF HEAT-CAPACITY AT NORMAL-INCOMMENSURATE PHASE-TRANSITION IN K2ZNCL4 AND K2SEO4

  H HAGA, R NOZAKI, Y SHIOZAKI, K EMA

  JOURNAL OF THE PHYSICAL SOCIETY OF JAPAN 64 11 4258 - 4264 1995年11月 [査読有り][通常論文]
   

  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.
• Three Dimensional XY Critical Behavior of Heat Capacity at Normal-Incommensurate Phase Transition in Rb2ZnCl4

  Hisashi Haga, Akira Onodera, Yoichi Shiozaki, Kenji Ema, Hideaki Sakata

  Journal of the Physical Society of Japan 64 3 822 - 829 1995年 [査読有り][通常論文]
   

  A high-resolution heat capacity measurement has been carried out near the normal-incommensurate phase transition in Rb2ZnCl4 by an ac calorimeter. The obtained data have been analyzed with a renormalization-group expression which includes correction terms up to the second order. It was found that the heat capacity anomaly is described well with the three dimensional XY model the critical exponent, the critical amplitude ratio, and the universal constant derived from the first-order correction amplitude agree well with the theoretically expected values. © 1995, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.
• A calorimetric study of RS1-xARSx mixed crystal system (0.9 ≤ x ≤ 1.0)

  Natsuko Noda, Hisashi Haga, Toshio Kikuta, Ryusuke Nozaki, Yoichi Shiozaki

  Ferroelectrics 168 1 169 - 175 1995年 [査読有り][通常論文]
   

  The specific heat in the region IV (0.9 ≤ x ≤ 1) of RS1-x-ARSx mixed crystal system has been measured between 80 K and 290 K by AC method. The anomaly of the specific heat has a large peak its apparent height increases with x. Thermal hysteresis between 0.5 K and 1.0 K for the anomalies were observed. This indicates that the phase transition seems to be of the first order. In many cases, the specific heat anomaly with two peaks was observed in the whole range of the region IV. The cause of this phenomenon also is discussed using the results of dielectric measurements. © 1995, Taylor & Francis Group, LLC. All rights reserved.
• Thermal and dielectric studies on ferroelectric (CH3NHCH2COOH)3CaCl2 (TSCC)

  Hisashi Haga, Akira Onodera, Haruyasu Yamashita, Yoichi Shiozaki

  Ferroelectrics 159 1 55 - 60 1994年09月01日 [査読有り][通常論文]
   

  Dielectric constants, birefringences, spontaneous polarization in TSCC were measured in a wide temperature range from 4.2 to 300 K. Dielectric constant along the b-axis exhibits an anomaly corresponding to a new phase transition at 65 K. D-E hysteresis loops were detected below 65 K: This indicates that the low-temperature phase is also ferroelectric. Changes in spontaneous polarization and birefringences were observed at 65 K. Anomalously large quadratic electrooptical coefficients were obtained. Rhodes-Wohlfarth ratio is estimated to be 1.5, which is not any typical value of order-disorder or displacive phase transition. Thermal behaviour around the ferroelectric phase transition at 130 K is in good agreement with the short-range behaviour in the temperature range of 3 K< |T -130 K|< 16 K. © 1994, Taylor & Francis Group, LLC. All rights reserved.
• SPECIFIC HEAT AND CRITICAL BEHAVIOR IN THIOUREA AT NORMAL-INC PHASE TRANSITION

  Akira Onodera, Hisashi Haga, Francoise Denoyer, Jean Godart, Yoichi Shiozaki

  FERROELECTRICS 155 305 - 310 1994年 [査読有り][通常論文]
   

  The specific heat of a type-II incommensurate ferroelectric SC(ND2)(2) was measured over a wide temperature region with an AC calorimeter. The critical index alpha associated with the normal-incommensurate phase transition is 0.12 +/- 0.02. This value does not coincide with alpha = 0 in the classical mean-field theory or with alpha = -0.007 in the 3d XY-model. It is rather close to that of the 3d Ising model. A crossover from a logarithmic region to a power-law region was found at reduced temperature t(cr) = 8x10(-3).
• KINETIC EVOLUTION IN THE INCOMMENSURATE PHASE IN K2ZnCl4

  Akira Onodera, Oto Watanabe, Haruyasu Yamashita, Hisashi Haga, Yoichi Shiozaki

  FERROELECTRICS 155 299 - 304 1994年 [査読有り][通常論文]
   

  Time evolution of dielectric constants in the incommensurate phase of K2ZnCl4 was observed. The dielectric constants showed a long-term relaxation when the sample was held at a constant temperature. They did not relax exponentially but obeyed a stretched exponential exp{-(t/tau)(beta)} with beta=0.6 and tau=22 min. at 436 K. This evidence indicates that the slow relaxation phenomenon can not be explained by the DDW theory, but is attributable to the kinetic process of nucleation and annihilation of discommensurations or to DDW diffusion with a wide distribution of relaxation times. No clear fine discontinuous stepwise changes in E were found even at the slow scanning speed of 0.001 K/min.
• DIELECTRIC BEHAVIOR AND RELAXATION PROCESS IN MODULATED PHASES IN THIOUREA

  Akira Onodera, Oto Watanabe, Haruyasu Yamashita, Hisashi Haga, Yoichi Shiozaki

  FERROELECTRICS 155 311 - 316 1994年 [査読有り][通常論文]
   

  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.
• Thermal and Dielectric Properties of a New Ferroelectric LaBGeO5

  Akira Onodera, Hisashi Haga, Boris A. Strukov, Alexander A. Belov, Sergei A. Taraskin, Haruyasu Yamashita, Yoshiaki Uesu

  Journal of the Physical Society of Japan 62 12 4311 - 4315 1993年 [査読有り][通常論文]
   

  Thermal and dielectric properties of LaBGeO2, a new ferroelectric with a still-wellite-type structure are investigated. The specific heat, Cp, shows one clear anomaly (J 5=0.114 R) at 802.5 K. The basic thermodynamic parameters are determined on the basis of the Landau theory. However, an additional hump in Cp was found in a different sample just above T^. This dependence on samples is confirmed by the measurements of dielectric constants. The appearance of the new intermediate phase may be caused by some impurities or defects. The Rhodes-Wohlfarth plot suggests that the nature of the phase transitions is of the displacive-type. © 1993, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.
• New Phase Transition in Ferroelectric (CH3NHCH2COOH)3CaCl2 at Low Temperatures

  Hisashi Haga, Akira Onodera, Yoichi Shiozaki, Haruyasu Yamashita

  Journal of the Physical Society of Japan 62 6 1857 - 1859 1993年 [査読有り][通常論文]
   

  The specific heat of ferroelectric (CH3NHCH2COOH)3CaCl2 has been measured precisely with an AC calorimeter. Two anomalies of specific heat are observed: one is associated with a ferroelectric phase transition at T1 = 130 K and the other is associated with a new phase transition at T2=65 K. This shows the existence of a new low-temperature phase, which probably corresponds to a high-pressure antiferroelectric phase reported previously. Transition entropies are estimated as 2.51 and 1.16 [J-mol-1 K-1] for phase transitions at T1 and T2, respectively. © 1993, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.
• Critical Behaviour of Specific Heat in Ferroelectric K2Z11CI4 and (NH4)2BeF4 at Normal-Incommensurate Phase Transition

  Hisashi Haga, Akira Onodera

  Journal of the Physical Society of Japan 62 5 1597 - 1602 1993年 [査読有り][通常論文]
   

  Measurements of specific heat were carried out on structurally incommensurate ferroelectrics K2ZnCl4 and (NH4)2BeF4 over a wide temperature region with an AC calorimetric technique. Critical indices a associated with the normal-incommensurate phase transition are obtained as 0.12±0.02 for K2ZnCl4 and 0.18±0.02 for (NH4)2 BeF4. These values do not agree with that in the 3d XY-model proposed by Bruce and Cowley, and are rather comparable with a=0.125 for the 3 d Ising-model. A crossover from a logarithmic behaviour to a power-law behaviour was found at reduced temperatures cr=l x 10-3 for K2ZnCl4 and 5 x 10-3 for (NH4)2BeF4, respectively. © 1993, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.
• Specific heat and critical indices associated with normal-incommensurate phase transition in K2ZnCl4 and (NH4)2BeF4

  Hisashi Haga, Akira Onodera, Yoichi Shiozaki

  Ferroelectrics 125 1 123 - 128 1992年01月01日 [査読有り][通常論文]
   

  Specific heat of ferroelectric k2ZnCI4 and (NH4)BeF4 which are type-I incommensurate compounds in the family of ammonium sulphate, was studied over a wide temperature region by using a. c. calorimetry method. Critical indices a associated with normal-incommensurate phase transition are 0.61 for K2ZnCI4 and 0.44 for (NH4)BeF4 above T1, which are quite different from that of typical ferroelectric triglycine sulphate. In both crystals, the anomalous specific heat shows a crossover from a logarithmic region to a power-law region at reduced temperatures tc=2x10-3 for k2ZnCI4 and 8x10-3 for (NH4)2BeF4. © 1992, Taylor & Francis Group, LLC. All rights reserved.
• Dielectric properties and phase transition in cesium tetrachlorozincate Cs2ZnCl4

  Akira Onodera, Oto Watanabe, Hisashi Haga, Toshio Kikuta, Eisuke Suzuki, Haruyasu Yamashita, Yoichi Shiozaki, Haruyasu Yamashita

  Ferroelectrics 125 1 141 - 146 1992年01月01日 [査読有り][通常論文]
   

  Dielectric and specific heat measurements of Cs2ZnCl4 single crystals were made in a wide temperature range. The temperature dependence of dielectric constants shows two anomalies at T(= 572 and T2=326 K. Specific heat and X-ray intensity of (8 0 0) exhibit also two corresponding anomalies, which are an evidence of structural phase transitions. Ferroelectric hysteresis loop has not been detected yet, but there exist at least two phase transitions. © 1992, Taylor & Francis Group, LLC. All rights reserved.

MISC

• ゲル基質上の上皮細胞シートが引き起こす浸透圧勾配依存的なドーム形成とその数理モデル

  秋山正和, 石原(石田)すみれ, 須志田隆道, 芳賀永 日本応用数理学会年会講演予稿集(CD-ROM) 2020 2020年
• 上皮細胞シートが引き起こす浸透圧勾配依存的なパターン形成とその数理モデル

  秋山正和, 石原(石田)すみれ, 須志田隆道, 芳賀永 日本数理生物学会年会(Web) 30th 2020年
• Fascin1により発現亢進されるLGR5は乳がんの浸潤を促進する(LGR5 up-regulated by Fascin1 promotes breast cancer cell invasion)

  伊東 祐紀, 石原 誠一郎, 加野 将之, 松原 久裕, 芳賀 永 日本癌学会総会記事 78回 P -3249 2019年09月
• 細胞・組織動態解析と数理モデルで解く形態形成の原理 細胞の集団運動と3次元形態形成に対する数理的アプローチ

  秋山 正和, 須志田 隆道, 森 瞳, 石田 すみれ, 芳賀 永 日本生化学会大会プログラム・講演要旨集 92回 [2S15a -07] 2019年09月 [査読無し][通常論文]
  
• 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年02月 [査読無し][通常論文]
  
• 基質の軟らかさが誘引する上皮細胞の集団運動と3次元形態形成

  芳賀 永 生物物理 58 (4) 196 -199 2018年 [査読無し][通常論文]
   

  <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>
• RANKLによるインテグリンα2の発現亢進はインテグリンβ2の細胞内輸送を介して細胞接着を亢進する

  大場雄介, 山田珠希, 山田珠希, 山田珠希, 津田真寿美, 藤岡容一朗, 藤岡真理, 堀内浩水, 堀口美香, 佐藤絢, ネパール ブラバ, 王せい, 柏木彩花, 西出真也, 南保明日香, 芳賀永, 田中伸哉, 進藤正信 日本生理学雑誌(Web) 79 (2) 49 (WEB ONLY) -49 2017年05月 [査読無し][通常論文]
  
• Fascin-1による転写因子Stat3を介したがん細胞の浸潤促進

  高橋 都史樹, 温田 晃弘, 加野 将之, 松原 久裕, 芳賀 永 日本癌学会総会記事 75回 P -1094 2016年10月
• 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年 [査読無し][通常論文]
  
• 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年12月 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 放射線により誘発されるがん細胞の再増殖 : 移植可能なマウス肉腫細胞を用いた実験から類推されるメカニズム

  西岡 健, 安田 元昭, 武島 嗣英, 芳賀 永, 宮井 優介, 柴田 健一郎, 山崎 理衣, 白土 博樹, 手塚 正博, 伊達 広行 北海道醫學雜誌 = Acta medica Hokkaidonensia 87 (4) 192 -192 2012年08月01日
• Regulatory mechanism of integrin beta 1 expression in collectively migrating cancer cells.

  T. Kato, A. Enomoto, H. Haga, S. Ishida, M. Asai, N. Asai, M. Takahashi MOLECULAR BIOLOGY OF THE CELL 23 2012年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• Involvement of cell polarity and cell migration in folding phenomena of epithelial sheets

  S. Ishida, R. Tanaka, N. Yamaguchi, T. Mizutani, K. Kawabata, H. Haga MOLECULAR BIOLOGY OF THE CELL 23 2012年 [査読無し][通常論文]
  
• 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年05月 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 走査型プローブ顕微鏡による細胞機能の解析

  水谷武臣, 芳賀永, 加藤幸作, 川端和重 細胞 40 (6) 250 -253 2008年06月20日 [査読無し][通常論文]
  
• 走査型プローブ顕微鏡を用いた生細胞表面の弾性分布測定

  芳賀 永, 水谷 武臣, 川端 和重 表面科学 = Journal of The Surface Science Society of Japan 29 (4) 235 -238 2008年04月10日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 30pXG-5 寒天ゲルの静止摩擦における温度効果(30pXG その他の系(パターン形成・ソフトマター・地震・実験),領域11(統計力学,物性基礎論,応用数学,力学,流体物理))

  稲月 剛久, 芳賀 永, 根本 幸児, 川端 和重 日本物理学会講演概要集 61 (1) 338 -338 2006年03月04日
• 広域走査型プローブ顕微鏡の開発と細胞生物学への応用

  水谷 武臣, 芳賀 永, 川端 和重 生物物理 45 (2) 105 -108 2005年03月25日 [査読無し][通常論文]
  
• 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年01月 [査読無し][通常論文]
  
• 細胞のダイナミクスと集団運動(ソフトマターの物理学2004-変形と流動-,研究会報告)

  水谷 武臣, 芳賀 永, 川端 和重 物性研究 83 (3) 333 -334 2004年12月20日 [査読無し][通常論文]
   

  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年11月 [査読無し][通常論文]
  
• 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年11月 [査読無し][通常論文]
  
• Collective and unidirectional movement of epithelial cells on a collagen gel substrate

  H Haga, K Hayashi, K Kawabata MOLECULAR BIOLOGY OF THE CELL 15 179A -179A 2004年11月 [査読無し][通常論文]
  
• 14aTC-9 寒天ゲルの静止摩擦の温度効果(統計力学・ソフトマター・摩擦, 領域 11)

  川端 和重, 加藤 秀章, 新田 高洋, 根本 幸児, 芳賀 永 日本物理学会講演概要集 59 (2) 232 -232 2004年08月25日
• 29pWA-4 高分子ゲルの静止摩擦 : 真実接触領域の構造の待機時間効果(ソフトマター(液晶・高分子・合金など))(領域11)

  加藤 秀章, 新田 高洋, 芳賀 永, 根本 幸児, 川端 和重 日本物理学会講演概要集 59 (1) 309 -309 2004年03月03日
• 高分子ゲルの静止摩擦の待機時間効果 : 真実接触領域の直接観察(摩擦の物理,研究会報告)

  加藤 秀章, 新田 高洋, 芳賀 永, 川端 和重 物性研究 81 (6) 860 -863 2004年
• 109 ナノ力学 SPM を用いた細胞運動に伴うかたさ分布の時間変化測定

  芳賀 永, 永山 昌史, 高橋 正行, 川端 和重 バイオエンジニアリング講演会講演論文集 2003 (15) 19 -20 2003年01月20日
• Cooperative movement of epithelial cells on soft collagen gel

  H Haga, N Hirakawa, T Iarahara, K Hayashi, M Takahashi, Y Kawabata MOLECULAR BIOLOGY OF THE CELL 13 78A -78A 2002年11月 [査読無し][通常論文]
  
• 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年11月 [査読無し][通常論文]
  
• 6aTC-8 高分子ゲルの滑り摩擦:静止摩擦力III(その他の系,領域11)

  新田 高洋, 川端 和重, 芳賀 永 日本物理学会講演概要集 57 (2) 211 -211 2002年08月13日
• ハイブリッド走査型プローブ顕微鏡による生細胞の力学的観察

  川端 和重, 佐戸 康洋, 永山 昌史, 芳賀 永 光学 31 (6) 483 -488 2002年06月10日
• 24pXK-13 高分子ゲルの滑り摩擦 : 静止摩擦力II(24pXK 生物・生態系,パターン形成・摩擦,領域11(統計力学,物性基礎論,応用数学,力学,流体物理分野))

  新田 高洋, 芳賀 永, 川端 和重 日本物理学会講演概要集 57 (1) 241 -241 2002年03月01日
• Stiffness change of living fibroblasts depending on cell motion

  M Nagayama, H Haga, K Kawabata MOLECULAR BIOLOGY OF THE CELL 12 171A -171A 2001年11月 [査読無し][通常論文]
  
• 919 水中に置かれた生体の原子間力顕微鏡を用いた粘弾性評価(GS-2 バイオエンジニアリング(2))

  田中 芳雄, 平井 義彦, 兜 雅章, 川端 和重, 芳賀 永, 永山 昌史 関西支部講演会講演論文集 2001 (76) "9 -43"-"9-44" 2001年03月16日 

  Knowledge of viscoelasticity distribution of a living biological cell is useful for investigation of a relationship between its motility and cellular structure. This paper proposes a strict evaluation method of viscoelasticity of cells through vibration analysis of AFM cantilever in water by considering resistance force of cells against cantilever tip penetration and drag force of water due to the cantilever vibration. Cyclic penetration of the tip is presently given by the vibration of the table, while additional vertical motion is applied to the table to avoid the effect of. topography on the evaluation of the viscoelasticity. A map of viscoelasticity distribution is taken by scanning motion of the table.
• AFMを用いた細胞運動に伴う粘弾性分布の時間変化測定

  永山 昌史, 芳賀 永, 川端 和重, 三本木 孝 電子顕微鏡 35 359 -359 2000年05月01日
• 23aZC-3 AFMによる寒天ゲルの局所弾性率マッピング

  新田 高洋, 芳賀 永, 川端 和重, 阿部 和厚, 三本木 孝 日本物理学会講演概要集 55 (1) 285 -285 2000年03月10日
• 液中環境における生きた細胞の粘弾性マッピング

  川端 和重, 芳賀 永 Molecular electronics and bioelectronics 10 (4) 215 -220 1999年10月29日
• 原子間力顕微鏡による生きた細胞の粘弾性測定(III)

  芳賀 永, 佐々木 茂雄, 新田 高洋, 川端 和重, 伊藤 悦朗, 阿部 和厚, 三本木 孝 生物物理 38 (2) S144 1998年09月07日
• 27p-Q-9 グリア細胞表面における細胞骨格を反映した微細構造のAFM観察

  山根 ゆか子, 畠山 大, 芳賀 永, 川端 和重, 阿部 和厚, 伊藤 悦朗 日本物理学会講演概要集 53 (2) 857 -857 1998年09月05日
• 1p-YB-3 AFMによる生きた繊維芽細胞の粘弾性測定II〜葉状仮足の微細構造と時間変化〜

  佐々木 茂雄, 芳賀 永, 川端 和重, 伊藤 悦朗, 阿部 和厚, 三本木 孝 日本物理学会講演概要集 53 (1) 737 -737 1998年03月10日
• 1p-YB-4 AFMによる生きた神経細胞の粘弾性測定III : 葉状仮足にみられる球状微細構造

  芳賀 永, 森本 真由美, 佐々木 茂雄, 川端 和重, 伊藤 悦朗, 阿部 和厚, 三本木 孝 日本物理学会講演概要集 53 (1) 738 -738 1998年03月10日
• 8a-P-4 原子間力顕微鏡を用いた生きた細胞の粘弾性測定

  芳賀 永, 森本 真由美, 佐々木 茂雄, 川端 和重, 伊藤 悦朗, 阿部 和厚, 三本木 孝 日本物理学会講演概要集 52 (2) 853 -853 1997年09月16日
• 29p-YD-7 ACカロリメトリーによる一次相転移の研究IV

  中野 秀彦, 野田 菜摘子, 芳賀 永, 野崎 龍介, 塩崎 洋一 日本物理学会講演概要集 52 (1) 138 -138 1997年03月17日
• 29p-YD-4 RS-ARS混晶の比熱IV

  野田 菜摘子, 中野 秀彦, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集 52 (1) 137 -137 1997年03月17日
• 3a-R-11 ACカロリメトリーによる一次相転移の研究III

  中野 秀彦, 野田 菜摘子, 芳賀 永, 野嵜 龍介, 塩[ザキ] 洋一 日本物理学会講演概要集. 年会 51 (2) 116 -116 1996年03月15日
• 3aーR-6 RS-ARS混晶系の比熱III

  野田 菜摘子, 中野 秀彦, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 年会 51 (2) 114 -114 1996年03月15日
• 28a-YD-11 RS-ARSの複素比熱

  中野 秀彦, 野田 菜摘子, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 秋の分科会 1995 (2) 95 -95 1995年09月12日
• 28a-A-11 ACカロリメトリーによる1次相転移の研究 II

  野田 菜摘子, 中野 秀彦, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 年会 50 (3) 673 -673 1995年03月16日
• 31a-YK-6 (RS)_<1-x>-(ARS)_x 混晶系0≤x<0.9 の比熱

  野田 菜摘子, 中野 秀彦, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 年会 50 (2) 101 -101 1995年03月16日
• 31a-YK-5 RS_<(1-x)>-ARS_x 混晶系(0.18

  土岐 裕亮, 中野 秀彦, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 年会 50 (2) 101 -101 1995年03月16日
• 2p-B-3 ACカロリメトリーによる1次相転移の研究

  野田 菜摘子, 中野 秀彦, 芳賀 永, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 秋の分科会 1994 (3) 544 -544 1994年08月16日
• 15p-J-8 (RS)_<1-X>-(ARS)_X混晶系0.9

  野田 菜摘子, 芳賀 永, 喜久田 寿郎, 野嵜 龍介, 塩崎 洋一 日本物理学会講演概要集. 秋の分科会 1993 (2) 122 -122 1993年09月20日

書籍等出版物

• 実験医学 増刊 『線維化 : 慢性疾患のキープロセス』 組織の「硬さ」から迫る線維症研究の新展開

  石原 誠一郎, 芳賀 永 (担当:共著範囲:第4章 第4節)
  羊土社 2020年08月 (ISBN: 9784758103886) 196p
• 三次元培養における培養手法と周辺技術動向

  芳賀 永 (担当:共著範囲:三次元培養法（ハイドロゲル法）)
  情報機構 2019年04月
• Chromosome Nanoscience and Technology

  TAYLOR & FRANCIS GROUP 2007年

担当経験のある科目（授業）

生物系の統計力学北海道大学理学部
生物系の量子力学北海道大学理学部
少人数討論型育成プログラム北海道大学大学院生命科学院

所属学協会

• 日本メカノバイオロジー学会   日本細胞生物学会   日本癌学会   

共同研究・競争的資金等の研究課題

• がん細胞に集団浸潤もたらす細胞間接着構造とシグナル経路の探索

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 芳賀 永

   

  多くのがん細胞種は細胞間の接着を維持したまま集団で浸潤する．このことを集団浸潤という．その機序については不明な点が多い．本研究は，がん細胞にみられる細胞間接着構造と細胞間に形成される仮足構造を観察・解析するとともに，それらの構成分子およびシグナル経路を明らかにすることで集団浸潤の普遍的機序に迫る．研究目的で掲げた3つの問いである①集団浸潤をもたらすシグナル経路の探索，②細胞間に特異的に見られる仮足構造の解析，③集団浸潤の普遍性の検証のうち，令和3年度では，①集団浸潤をもたらすシグナル経路の探索を行った． ヒト皮膚扁平上皮がん細胞株（A431細胞）から高浸潤と低浸潤のサブクローン株を樹立し，DNAマイクロアレイによってサブクローン間の発現遺伝子を比較したところ，これまでにkeratin-1，10，14，JAK-STAT経路などの遺伝子発現に有意な差があることが明らかとなっていた．そこで，転写因子であるSTAT1に着目し，RNA干渉法によってSTAT1の発現を抑制したところ，高浸潤サブクローン株の集団浸潤が有意に抑制されるという結果を得た．さらに，STAT1を活性化させるサイトカインであるインターフェロンβ（INFB）の発現と局在を免疫蛍光染色法によって観察したところ，高浸潤サブクローン株では細胞-細胞間にINFBが局在する一方，低浸潤サブクローン株では細胞-細胞間での局在は観察されなかった．加えて，高浸潤サブクローン株でINFBの発現をノックダウンしたところ，STAT1は活性化せず，集団浸潤も抑制されることが明らかとなった．さらに，EGTAを用いて細胞-細胞間接着を緩めたところ，高浸潤サブクローン株におけるINFBの細胞-細胞間局在が消失し，集団浸潤が抑制された．これらの結果は，IFNBが細胞間隙に蓄積しSTAT1が活性化することで集団浸潤が誘引されることを示している．
• 足場材の硬さの違いを利用した上皮角化・非角化様式解明と培養口腔粘膜作成法への応用

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 泉 健次, 芳賀 永, 石原 誠一郎, 加来 賢, 佐藤 大祐, 鈴木 絢子

   

  今年度は、コロナの影響で魚うろこコラーゲンの入手がままならず、既製品の魚うろこコラーゲンであるセルキャンパスしか手に入らなかったため、コラーゲンゲルの足場材の硬さをランダムに変えての培養口腔粘膜作成はできなかった。その分、セルキャンパスを用いて、コントロールの硬い培養皿面、同表面でカルシウム濃度をあげた分化培地、および、いわゆるコラーゲンゲルの軟性面で培養したケラチノサイトに対し、運動能解析と外注によるマイクロアレイ解析による網羅的遺伝子発現分析（現在までに2サンプル解析終了し、現在もう2サンプル培養しており、外注予定）を実施し、結果がでたら新たに分担者に加わっていただいた凌先生にヒートマップとクラスター図を作成してもらい、ビッグデータ解析をお願いする。 細胞運動能解析では驚いたことに、コラーゲンゲル上の細胞運動能が、硬い培養皿面での細胞運動能より上回っていた。これは、いわゆる癌細胞のメカノバイオロジーと真逆の現象である。さらに、運動能が高いにも関わらず、ケラチノサイトの分化マーカー遺伝子発現が更新していることが示唆され、申請者の以前の基盤Ｂ研究で報告した結果とも相反するデータを得ており、今後考察を加え、メカニズム解明したい。また、コロナの影響で、研究分担者のいる北海道大学にＡＦＭを用いた細胞の硬さ検索ができなかった。
• がん細胞に集団浸潤を誘引する接触追従の機序解明と普遍性の検証

  文部科学省：学術研究助成基金助成金／基盤研究（Ｃ）

  研究期間 : 2017年 -2019年 

  代表者 : 芳賀 永
• インビトロ系における細胞シートからの３Ｄ形態形成

  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))

  研究期間 : 2015年 -2019年 

  代表者 : 芳賀 永
• ３Ｄ形態ロジックの国際共同研究を加速するバーチャル研究所

  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))

  研究期間 : 2015年 -2019年 

  代表者 : 近藤 滋
• 生物の３Ｄ形態を構築するロジック

  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))

  研究期間 : 2015年 -2019年 

  代表者 : 近藤 滋
• 細胞外基質の硬さに誘引される転写調節因子ATF5の活性と浸潤能亢進の機序

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2014年 -2016年 

  代表者 : 芳賀 永

   

  本研究では、基質の硬さが誘引するがん細胞の浸潤能との関係を調べ、がん細胞が悪性化する機序の解明を目指した。その結果、基質の硬さによって転写因子ATF5が活性化し、基質接着タンパク質であるインテグリンの発現に関与することでがん細胞の浸潤能が亢進することを明らかとした。さらに、がん細胞株を硬さの異なる基質中で培養したところ、硬い基質上では、ATF5が核内に移行する現象を見出した。加えて、ATF5の欠失変異体をがん細胞に発現させ、核内移行の機序を調べたところ、核外搬出シグナル（NES）が関与していることが明らかとなった。
• がん細胞は自身の生存をかけ放射線域を逃れるべく上皮間葉移行をなしうるか

  日本学術振興会：科学研究費助成事業

  研究期間 : 2012年04月 -2015年03月 

  代表者 : 西岡 健, 芳賀 永, 安田 元昭, 武島 嗣英

   

  今年度の実験において我々は5'UTRおよび3'UTR依存性発現調節の候補遺伝子の機能解析を行った。5'UTR結合タンパク質に関してはSRSFファミリーが有望であると考えられた。これらタンパク質はHIF-laあるいはcMYCの5'UTRを挿入したルシフェラーゼリポーターからのタンパク質発現を数倍に活性化することができた。これら候補遺伝子の発現を、マイクロアレイにて比較したところ、予想通り高転移性株では発現が高い傾向が認められた。これらの発現が遺伝子プロモーター領域の配列の違いによるものか否かを判断するため、アレイ解析に用いた細胞のゲノムDNAを出発材料にしてプロモーター領域のシークエンスを行ったところ顕著な塩基置換は認められず、メチル化などのエピジェネティックな変化の解析が今後の課題として提起された。3'UTR依存性発現調節の候補遺伝子としてELAVLファミリータンパク質の1つであるELAVL2の機能解析を行った。ELAVL1はほぼ全ての細胞において多量に発現し主に核局在を示すが、ELAVL2は主に細胞質に局在する。ELAVL2導入細胞株ではELAVL1を導入した細胞よりも、ARE mRNA由来のタンパク質発現レベルが有意に上昇していた。蛍光タンパク質タグを付与したHuRを単独で強制発現させてもELAVL1は核局在を示したが、ELAVL1とELAVL2を同時に発現させた細胞では細胞質局在するELAVL1が認められた。two-hybrid法にて、ELAVL1どうしの結合よりELAVL1-ELAVL2間のほうがより強い結合であることが示された。この結合にはELAVL2のN末端、C末端領域が必要であった。ELAVL1の核から細胞質への局在変化にはELAVL2が大きく関わっていること、ELAVL2の発現量はがん細胞の悪性度と相関することが示唆された。これまで3'UTR依存性の翻訳抑制機構を担うと報告されてきたELAVL1はむしろHIF-1αの翻訳効率を低下させることが示され、従来考えられてきた調節機構はより複雑なものであることが示唆された。
• 張力によるアクチンの構造変化と、それに依存したミオシンの結合増加および局在制御

  日本学術振興会：科学研究費助成事業

  研究期間 : 2012年04月 -2015年03月 

  代表者 : 上田 太郎, 祐村 恵彦, 芳賀 永, 野口 太郎, 安達 泰治

   

  真核細胞においてさまざまな重要現象に中心的に関与するアクチンフィラメントの機能分化機構を解明するため、アクチンフィラメントに張力が負荷されるとミオシンIIの結合が促進されるという仮説の検証を試みた。その結果、アメーバ細胞内のフィラメントに負荷される張力を見積もることができた。そうした張力をin vitroでフィラメントに負荷しミオシンIIの結合量の変化を見る実験に関しては、概ね実験系を確立でき、今後の測定への展望が開けた。蛍光エネルギー移動法による観察も最終的な結論を得るに至っていないが、細胞内のフィラメントに構造多型性があることが確認できた。
• ゲル基質の軟らかさと粘性を利用した上皮シートの3次元組織構築

  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))

  研究期間 : 2014年 -2015年 

  代表者 : 芳賀 永

   

  本研究課題は，ゲル基質を用いて上皮細胞に腸管絨毛のような立体構造（チューリップハット様の構造）を形成させるシステムを構築することを目的とする．研究代表者はこれまでに，上皮細胞をマトリゲル上に播種すると，上皮シートの辺縁部が内側に収縮し，ゲル基質を変形させながら回転運動を行い，絨毛構造を形成することを発見した．本研究では，この現象を組織再生の基礎技術とするために，①上皮シートの辺縁部が収縮する機構，②ゲル基質の粘性がもたらす効果，③シート全体が回転運動する機構の3点に焦点を当てて研究を遂行した． 平成27年度では，交付申請書における研究実施計画に従って，リーダー細胞の出現による形態形成への機序解明と様々な粘性係数をもつ粘性基質の開発を行った．実験の結果，上皮間葉転移（EMT）を誘引する受容体を薬剤阻害してもチューリップハット構造の形成が阻害されることはなかった．リーダー細胞の出現，およびチューリップハット構造の形成にはEMTのシグナル経路は関与しないことが明らかとなった．また，粘性基質の開発については，シリコーンオイルにPDMSを混ぜることで任意の粘性係数を実現することに成功した．粘性基質上に播種した上皮細胞が様々な形の突起構造，管状構造を形成することを発見した．さらに，従来法であるマトリゲルに架橋剤としてゲニピンを混ぜることで，任意の粘性係数をもつ新規のマトリゲル基質を開発した．ゲニピンの濃度を調節し，粘性係数を変えことで任意のサイズの突起構造を形成させることに成功した． これらの結果は，平成27年度内にScientific Reports誌に原著論文として公開することができた．
• 多数の細胞の力と運動がもたらす管腔構造形成のロジック

  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))

  研究期間 : 2013年 -2014年 

  代表者 : 芳賀 永

   

  本研究課題は，上皮細胞の集団が協調して力を出し合い，管腔組織を形成するロジックを明らかにすることを目的とする．研究代表者はこれまでに，上皮細胞（MDCK細胞）をコラーゲンゲルで重層すると，上皮シートの辺縁部が内側に収縮し，シート全体が湾曲しながら折り返されることで管腔構造が形成されることを発見した．本研究では，この現象が生じるロジックを解明するために，①上皮シート端が折り返す機構，②折り返した細胞に追随して後続細胞が集団で運動する機構，③上皮シートが湾曲し間隙が形成される機構の3点に焦点を当てて研究を遂行した． 平成26年度では，交付申請書における研究実施計画に従って，上皮シート辺縁部を取り囲むアクトミオシン束の収縮力発生機構について調べた．とくに，細胞に大きな収縮力をもたらすII型ミオシン調節軽鎖（MRLC）の2重リン酸化機構に着目して実験を行った．具体的には，阻害剤投与，免疫蛍光染色，siRNAによる発現抑制，FRETプローブを用いた蛍光ライブイメージングを行うことで，MRLCの2重リン酸化に寄与するリン酸化酵素（ROCK，ZIP-kinase，MLCK）の働きについて検討した．その結果，MLCKは折り返しには寄与せず，ROCKとZIP-kinaseの寄与が大きいことが明らかとなった．とくに，ZIP-kinaseの働きについてはあまり知られていなかったので，今回の発見は細胞の集団運動を理解する上で意義が大きいといえる．さらに，研究実施計画に従って，MMP-8の分泌と管腔形成との関係についても調べた．MMP-8の阻害剤を投与したところ，管腔形成に大きな影響を及ぼさないことが明らかとなった． これらの結果は，平成26年度内にPLoS ONE誌に原著論文として公開することができた．
• 動体追跡医科学研究

  文部科学省：科学研究費補助金(基盤研究(A))

  研究期間 : 2009年 -2012年 

  代表者 : 白土 博樹, 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...
• 基質の軟らかさによって誘引される細胞の協調的集団運動に関する研究

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2009年 -2011年 

  代表者 : 芳賀 永, 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...
• 力学的応答測定を利用した極微小タンパク質結晶の判別・評価法の開発

  文部科学省：科学研究費補助金(挑戦的萌芽研究)

  研究期間 : 2009年 -2010年 

  代表者 : 田中 勲, 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...
• 放射線照射を生き延びた癌細胞に秘められた謎:癌根絶への多角的アプローチ

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2008年 -2010年 

  代表者 : 西岡 健, 芳賀 永, 安田 元昭, 本間 明宏, 堤香 織, 酒井 正春

   

  放射線後生き残った細胞(以下IRとをす)の遺伝子発現をcDNAアレイにより調べた。マウス線維肉腫細胞のセルラインQRsPを使用し10Gyの照射を施行した。コロニーアッセイとともに6つのクローンの樹立が行われた。照射を生き延びた細胞では、サイクリン依存性キナーゼ阻害剤(CKI)、p16 p57 の著明な発現低下;14.8および12.0の低下、また耐性クローン(IR)をマウスに移植したところ移植比100%がえられた。この細胞株にCD34がふくまれていることをかんがみると、がん幹細胞が放射線耐性に含まれていた可能性がある。
• 細胞コロニー内の細胞に個別に蓄積される力学的記憶の解明

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2006年 -2008年 

  代表者 : 川端 和重, 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。
• 放射線治療中に生ずる高度増殖浸潤能クローンの封じ込め

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2006年 -2007年 

  代表者 : 西岡 健, 安田 元昭, 芳賀 永, 本間 明宏, 堤 香織

   

  マウス繊維芽肉腫細胞株QR s P細胞に10Gyの放射線照射を施行し、生存したコロニーから24株をクローニングした。これらに対しさらに10Gy照射したところコロニーの出現のしかたはまちまちであり、母集団の中には放射線高感受性のものもあれば抵抗性のものもあることが判明した。このうち最も抵抗性の株(10-1IRと命名)と最も高感受性の株10-5IRについて様々な角度から相違点を見つけ出した。まず親株との間でそれぞれc DNAアレイを行った。またIR群それぞれをC57BL/6マウスに細胞移植を行ったところ造腫瘍能には変わりがないことが判明した。この腫瘍から細胞株を再び作成し親株との間で2回目のc DNAアレイを施行した。この計4回のアレイデータから、それぞれ親株と似ているもの、異なるものを抽出し、さらにそれらの遺伝子群について10-1IRと10-5IRの間で異なる遺伝子を70個検出した。このなかには転写因子であるATF5が存在した。放射線照射をmimicするためp53を強制導入して親株と10-5IRの細胞死分画をフローサイトメーターみたところ、親株で7%、10-5IRで25%と明らかに10-5IR細胞においてp53によるアポトーシスが多く見られた。次にATF5を10-5IRに強制導入しコロニーアッセイによる10Gy照射後の生存コロニー数、フローサイトメーターによるアポトーシスの文画...
• 細胞システムにおける「力学平衡」モデルの実証

  文部科学省：科学研究費補助金(萌芽研究)

  研究期間 : 2003年 -2004年 

  代表者 : 川端 和重, 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...
• ナノ力学プローブ顕微鏡による結合組織の形成過程における力学的制御機構の解明

  文部科学省：科学研究費補助金(若手研究(B))

  研究期間 : 2002年 -2004年 

  代表者 : 芳賀 永

   

  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。
• 生きた動物細胞の粘弾性分布の走査型プローブ顕微鏡による定量的評価法の開発

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2002年 -2003年 

  代表者 : 平井 義彦, 芳賀 永, 川端 和重, 田中 芳雄, 川田 博昭, 菊田 久雄

   

  細胞のマイクロ粘弾性解析手法として,探針をコンタクトモードで走査しながら試料に周期的な応力を加え、たわみ信号に現れる周期的な応答から試料の粘弾性を評価フォースモジュレーション法について,試料の粘弾性と溶液の粘性の両方を考慮に入れたカンチレバーにおける1次元の振動方程式を導入し,この方程式に基づいた数値計算により、カンチレバー振動の振幅・位相から、Hertzモデルを複素弾性率に拡張し、1次元の弾性[N/m]と粘性[Ns/m]から弾性率と粘性係数を計算した。次に,走査型プローブ顕微鏡(SPM)のカンチレバー探針を,試料に周期的に押込むときに,特に,探針が試料と接触・乖離を繰返す場合につき、カンチレバー運動に対する流体の抗力を考慮した高次の振動モードを含むカンチレバー運動方程式を解析的に解き,カンチレバー根元の押込み方向励振振動に対するSPMカンチレバー先端の振動振幅比,位相遅れと,試料の表面剛性,表面粘性との関係を導出し,粘弾性分布を定量的に評価する方法を構築した.これより,カンチレバーの位相差,振幅比と,試料の表面粘性,表面剛性の関係を示す粘弾性分布図を求めた.これを基にして,生きた細胞の粘弾性特性を測定するためのソフトウエアを開発した.これにもとづき,AFMからの測定データより,生きた細胞の粘弾性分布を測定するシステムの構築と,その有効性を検証した.
• 生細胞の組織形成過程における力学的制御機構の解明

  研究期間 : 1996年

産業財産権

• 特開2023-110368:細胞の培養方法、細胞の培養方法を用いたスクリーニング方法、培養装置及び培養キット  2023年08月09日

  石原 誠一郎, 芳賀 永, 石原 すみれ  国立大学法人北海道大学  202303008233677554
• 特開2019-123845:ゲルの製造方法及びコラーゲンゲル  2019年07月25日

社会貢献活動

• 全国大学生活協同組合連合会 理事（北海道ブロック運営委員長）

  期間 : 2022年01月 - 現在

  役割 : その他
• 北海道大学生活協同組合 監事会議長

  期間 : 2021年05月 - 現在

  役割 : その他