2012 Fiscal Year Final Research Report
Live Imaging for Two-photon microscopy: Membrane repair in Squid Gian Axon.
| Project/Area Number |
22659037
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Kagawa University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Toru 城西大学, 薬学部, 准教授 (60217049)
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| 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)
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| Project Period (FY) |
2010 – 2012
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| Keywords | 細胞機能形態学(細胞膜修復) |
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| 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.
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[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.
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Journal Title
PLoS One.
Volume: 8(4)
Pages: e61540
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[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
Year and Date
20130202-06
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