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Live Imaging for Two-photon microscopy: Membrane repair in Squid Gian Axon.

Research Project

Project/Area Number 22659037
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeSingle-year Grants
Research Field General anatomy (including Histology/Embryology)
Research InstitutionKagawa University

Principal Investigator

MIYAKE Katsuya  香川大学, 医学部, 准教授 (30219745)

Co-Investigator(Kenkyū-buntansha) TANAKA Toru  城西大学, 薬学部, 准教授 (60217049)
Co-Investigator(Renkei-kenkyūsha) ARAKI Shinichi  香川大学, 医学部, 教授 (10202748)
EGAMI Youhei  香川大学, 医学部, 助教 (80432780)
MATSUDA Chie  独立行政法人産業技術総合研究所, バイオメディカル部門, 主任研究員 (50344099)
FUKAI Naomi  奥羽大学, 歯学部, 教授 (60134681)
KANAGAWA Motoi  神戸大学, 医学研究科, 助教 (00448044)
Project Period (FY) 2010 – 2012
Project Status Completed (Fiscal Year 2012)
Budget Amount *help
¥2,990,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥390,000)
Fiscal Year 2012: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2011: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,300,000)
Keywords細胞機能形態学(細胞膜修復) / 細胞膜修復 / 細胞膜損傷 / 膜融合 / 二光子レーザー / イカ巨大神経 / 小胞輸送 / MICAL / アネキシン / 細胞膜修復装置 / カルシウム / 小胞融合
Research Abstract

Plasma membrane disruption is a common form of cell injury in mammalian tissues under physiological conditions. Cell survival depends on the initiation of a rapid (second time-scale) resealing response that is mounted only in the presence of physiological levels of extracellular Ca2+. Vesicle-vesicle and vesicle-plasma membrane fusion events occurring in cortical cytoplasm surrounding the defect are thought to be a crucial element of the resealing mechanism. Axolemmal repair has been also studied extensively in invertebrate giant axons because their large size facilitates the use of many techniques. However, it is not clear how to repair axolemmal injury which usually takes minutes to hours time scale. Therefore, we observed how to reseal axolemmal disruption with several fluorescent probes, FM dye, Calcein-AM, Rhodamin or FITC-Dextran(RhDx, FDx, 10kD) and fluo4-AM using multi-photon microscope. First , we examined individual narrow axons from the squid fin nerve bundle. FM1-43 was loaded by the cut ends of fin nerves to show internal vesicles, and then wounded the axolemma by two photon laser. The FM1-43-positive vesicles were recruited the way of the membrane -membrane contacts leading to the homotypic and very rapid (second time scale) exocytic fusion events required for membrane repair in the narrow fin nerve. Whereas, FM 1-43 or Ca2+by fluo4-AM did not get into the axoplasm of the giant axon through the axolemmal disruption. Furthermore, when natural sea water containing FDx (10kD) was injected, the fluorescence spread throughout the axoplasm. These results suggest that high Ca2+did not cause rapid fusion of intracellular membranes, creating a boundary that prevents spreading of FDx throughout the cytoplasm. We propose that the slow speed of calcium diffusion through axoplasm density leads to the slow axolemmal repair.

Report

(4 results)
  • 2012 Annual Research Report   Final Research Report ( PDF )
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • Research Products

    (38 results)

All 2013 2012 2011 2010 Other

All Journal Article (8 results) (of which Peer Reviewed: 4 results) Presentation (30 results)

  • [Journal Article] Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro.2013

    • Author(s)
      Tanaka A, Woltjen K, Miyake K, Hotta A, Ikeya M, Yamamoto T, Nishino T, Shoji E, Sehara-Fujisawa A, Manabe Y, Fujii N, Hanaoka K, Era T, Yamashita S, Isobe K, Kimura E, Sakurai H.
    • Journal Title

      PLoS One.

      Volume: 8(4)

    • NAID

      120005244565

    • Related Report
      2012 Final Research Report
  • [Journal Article] Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro.2013

    • Author(s)
      Tanaka, A., et al
    • Journal Title

      PLoS ONE

      Volume: 8(4) Issue: 4 Pages: e61540-e61540

    • DOI

      10.1371/journal.pone.0061540

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The C2A domain in dysferlin is important for association with MG53(TRIM72).2012

    • Author(s)
      Matsuda C, Miyake K, Kameyama K, Keduka E, Takeshima H, Imamura T, Araki N, Nishino I, Hayashi Y.
    • Journal Title

      PloS Curr.

      Volume: 4

    • Related Report
      2012 Final Research Report
  • [Journal Article] The C2A domain in dysferlin is important for association with MG53 (TRIM72).2012

    • Author(s)
      Chie Matsuda
    • Journal Title

      PLoS Curr.

      Volume: 4

    • DOI

      10.1371/5035add8caff4

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 高感度多光子顕微鏡による細胞膜修復のLIVEイメージング2011

    • Author(s)
      三宅克也、荒木伸一
    • Journal Title

      顕微鏡

      Volume: 46 Pages: 109-112

    • Related Report
      2011 Annual Research Report
  • [Journal Article] Micromorphology of resin-dentin interfaces using one-bottle etch&rinse and self-etching adhesive systems on laser-treated dentin surfaces : a confocal laser scanning microscope analysis.2010

    • Author(s)
      de Oliveira MT
    • Journal Title

      Lasers Surg Med.

      Volume: 42(7) Pages: 662-670

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer.2010

    • Author(s)
      Lostal W.
    • Journal Title

      Hum Mol Genet.

      Volume: 19(10) Pages: 1897-1907

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 高感度多光子顕微鏡による細胞膜修復のLIVE イメージング

    • Author(s)
      三宅克也、荒木伸一
    • Journal Title

      顕微鏡

      Volume: vol.46 Pages: 109-112

    • Related Report
      2012 Final Research Report
  • [Presentation] GaAsP搭載型多光子レーザー顕微鏡によるLIVEイメージング:細胞膜修復時におけるアネキシン結合タンパクS100A11の動態2013

    • Author(s)
      〓田 萌, 三宅克也, 松田知栄, 川合克久, 江上洋平, 林 由起子, 荒木伸一
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      香川
    • Related Report
      2012 Final Research Report
  • [Presentation] 多光子レーザー顕微鏡によるLIVEイメージング:細胞膜修復時におけるアネキシンA4の動態2013

    • Author(s)
      松尾巴瑠奈, 三宅克也,塚本真由,濱田萌,松田知栄,川合克久,江上洋平,林 由起子,荒木伸一
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      香川
    • Related Report
      2012 Final Research Report
  • [Presentation] 多光子レーザー顕微鏡によるLIVEイメージング:細胞膜修復時におけるアネキシンA7の動態2013

    • Author(s)
      塚本真由,三宅克也,松尾巴瑠奈,濱田萌,松田知栄,川合克久,江上洋平,林 由起子,荒木伸一
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      香川
    • Related Report
      2012 Final Research Report
  • [Presentation] GaAsP搭載型多光子レーザー顕微鏡によるLIVEイメージング:イカ神経線維の細胞膜修復2013

    • Author(s)
      三宅克也,H.M.Fishman, H.C.Pant, J.Zimmerberg, 荒木伸一
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      香川
    • Related Report
      2012 Final Research Report
  • [Presentation] MICAL1 an F-actin-disassembly factor links membrane repair2013

    • Author(s)
      Miyake K., Egami Y., Matsuda C., Hamada M., Hayashi Y., McNeil P.L., Araki N
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      香川
    • Related Report
      2012 Final Research Report
  • [Presentation] Requirement for cholesterol in membrane repair indicates a possible mechanism by which statins cause muscle injury.2013

    • Author(s)
      Miyake K., Tanaka T., McNeil P.L., Araki N.
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      香川
    • Related Report
      2012 Final Research Report
  • [Presentation] Multiple Antibody Co-localization Imaging of Skeletal Muscle.2013

    • Author(s)
      M. Ghochani, G. Humphrey, J. Farrington, K. Miyake, K. Micheva, B. Busse, P. Blank, S. Smith, J. Zimmerberg
    • Organizer
      Biophysical Society 57th Annual Meeting
    • Place of Presentation
      Phyladelphia, PA, USA
    • Related Report
      2012 Final Research Report
  • [Presentation] 多光子レーザー顕微鏡によるLIVEイメージング:細胞膜修復時におけるアネキシン結合タンパクS100A11の動態2013

    • Author(s)
      濵田萌
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      高松
    • Related Report
      2012 Annual Research Report
  • [Presentation] GaAsP搭載型多光子レーザー顕微鏡によるLIVEイメージング:イカ神経線維の細胞膜修復2013

    • Author(s)
      三宅克也
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      高松
    • Related Report
      2012 Annual Research Report
  • [Presentation] 多光子レーザー顕微鏡によるLIVEイメージング:細胞膜修復時におけるアネキシンA4の動態2013

    • Author(s)
      松尾巴瑠奈
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      高松
    • Related Report
      2012 Annual Research Report
  • [Presentation] Requirement for cholesterol in membrane repair indicates a possible mechanism by which statins cause muscle injury.2013

    • Author(s)
      Katsuya Miyake
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      高松
    • Related Report
      2012 Annual Research Report
  • [Presentation] 多光子レーザー顕微鏡によるLIVEイメージング:細胞膜修復時におけるアネキシンA7の動態2013

    • Author(s)
      塚本真由
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      高松
    • Related Report
      2012 Annual Research Report
  • [Presentation] MICAL1 an F-actin-disassembly factor links membrane repair.2013

    • Author(s)
      Katsuya Miyake
    • Organizer
      第118回日本解剖学会総会・全国学術集会
    • Place of Presentation
      高松
    • Related Report
      2012 Annual Research Report
  • [Presentation] Dysferlin and affixin is involved in sarcolemmal repair.2012

    • Author(s)
      Matsuda C, Miyake K, Kameyama K, Araki N, Nishino I and Hayashi Y.
    • Organizer
      17th International congress of the World Muscle Society.
    • Place of Presentation
      Perth, Australia
    • Year and Date
      2012-10-10
    • Related Report
      2012 Final Research Report
  • [Presentation] Dysferlin and affixin in sarcolemmal repair.2012

    • Author(s)
      Matsuda C, Miyake K, Kameyama K, Araki N, Nishino I and Hayashi Y.
    • Organizer
      New Directions in Biology and Disease of Skeletal Muscle Conference.
    • Place of Presentation
      New Orleans, LA, USA
    • Year and Date
      2012-06-18
    • Related Report
      2012 Final Research Report
  • [Presentation] GaAsP搭載型多光子レーザー顕微鏡によるLIVEイメージング:アネキシン小胞による細胞膜修復2012

    • Author(s)
      三宅克也, 松田知栄, 江上洋平,濱田 萌,林 由起子, 荒木伸一
    • Organizer
      第117回日本解剖学会総会・全国学術集会
    • Place of Presentation
      甲府
    • Related Report
      2012 Final Research Report
  • [Presentation] GaAsP 搭載型多光子レーザー顕微鏡による LIVE イメージング:イカ神経線維の細胞膜修復2012

    • Author(s)
      三宅克也,H.M.Fishman, H.C.Pant, J.Zimmerberg, 荒木伸一
    • Organizer
      日本解剖学会,第67回中国・四国支部学術集会
    • Place of Presentation
      宇部
    • Related Report
      2012 Final Research Report
  • [Presentation] 多光子レーザー顕微鏡による LIVE イメージング:アネキシン A4 およびA7 小胞による細胞膜修復2012

    • Author(s)
      松尾巴瑠奈,塚本真由,濱田 萌,三宅克也, 松田知栄,川合克久, 江上洋平,林 由起子,荒木伸一
    • Organizer
      日本解剖学会,第67回中国・四国支部学術集会
    • Place of Presentation
      宇部
    • Related Report
      2012 Final Research Report
  • [Presentation] MICAL1 an F-actin-disassembly factor links membrane repair.2012

    • Author(s)
      Miyake K., Egami Y., Matsuda C., Hamada M., McNeil P.L., Araki N.
    • Organizer
      America Society for Cell Biology, Annual Meeting
    • Place of Presentation
      San Francisco, CA, USA
    • Related Report
      2012 Final Research Report
  • [Presentation] 多光子レーザー顕微鏡によるLIVEイメージング:アネキシンA4およびA7小胞による細胞膜修復2012

    • Author(s)
      松尾巴瑠奈
    • Organizer
      日本解剖学会,第67回中国・四国支部学術集会
    • Place of Presentation
      宇部
    • Related Report
      2012 Annual Research Report
  • [Presentation] GaAsP搭載型多光子レーザー顕微鏡によるLIVEイメージング:イカ神経線維の細胞膜修復2012

    • Author(s)
      三宅克也
    • Organizer
      日本解剖学会,第67回中国・四国支部学術集会
    • Place of Presentation
      宇部
    • Related Report
      2012 Annual Research Report
  • [Presentation] MICAL1 an F-actin-disassembly factor links membrane repair.2012

    • Author(s)
      Katsuya Miyake
    • Organizer
      The American Society fro Cell Biology
    • Place of Presentation
      San Francisco, CA, USA
    • Related Report
      2012 Annual Research Report
  • [Presentation] GaAsP搭載型多光子レーザー顕微鏡によるLIVEイメージング:アネキシン小胞による細胞膜修復2012

    • Author(s)
      三宅, ら
    • Organizer
      日本解剖学会総会
    • Place of Presentation
      甲府
    • Related Report
      2011 Annual Research Report
  • [Presentation] Real time analysis of sarcolemmal repair mediated by dysferlin and MG53.2011

    • Author(s)
      Matsuda C., K Miyake, K Kameyama, H Takeshima, E Keduka, N Araki, I Nishino, YK Hayashi
    • Organizer
      16th International WMS Congress
    • Place of Presentation
      Algarve, Portugal
    • Year and Date
      2011-10-19
    • Related Report
      2012 Final Research Report
  • [Presentation] MICAL an F-actin-disassembly factor links membrane repair.2011

    • Author(s)
      Miyake K., Egami Y., McNeil P.L., Araki N
    • Organizer
      J.Physiol.Sci.
    • Related Report
      2012 Final Research Report
  • [Presentation] 高感度多光子顕微鏡による細胞膜修復の LIVE イメージング2011

    • Author(s)
      三宅克也, 松田知栄, 江上洋平,濱田 萌,林 由起子,荒木伸一
    • Organizer
      第66回中国・四国支部学術集会
    • Place of Presentation
      徳島
    • Related Report
      2012 Final Research Report
  • [Presentation] MICAL an F-actin-disassembly factor links membrane repair2011

    • Author(s)
      Katsuya Miyake
    • Organizer
      Fifth Annual Dysferlin Conference
    • Place of Presentation
      Chicago Illinois, U.S.A.
    • Related Report
      2011 Annual Research Report
  • [Presentation] 高感度多光子顕微鏡による細胞膜修復のLIVEイメージング2011

    • Author(s)
      三宅克也, 荒木伸一
    • Organizer
      顕微鏡学会第55回シンポジウム
    • Place of Presentation
      高松
    • Related Report
      2011 Annual Research Report
  • [Presentation] 高感度多光子顕微鏡による細胞膜修復のLIVEイメージング2011

    • Author(s)
      三宅克也, ら
    • Organizer
      日本解剖学会,第66回中国・四国支部学術集会
    • Place of Presentation
      徳島
    • Related Report
      2011 Annual Research Report
  • [Presentation] MICAL an F-actin-disassembly factor links membrane repair.2011

    • Author(s)
      Katsuya Myake
    • Organizer
      Physiol.Sci.,61(Suppl.1),s271,2011
    • Place of Presentation
      Yokohama, Japan
    • Related Report
      2010 Annual Research Report

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Published: 2010-08-23   Modified: 2019-07-29  

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