| Project/Area Number |
03404074
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Kyoto University |
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Principal Investigator |
ITO Tadanao (1993) Kyoto lniw, Suonce Associate Professor, 理学部, 助教授 (90093187)
大西 俊一 (1991-1992) 京都大学, 理学部, 教授 (00025272)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Satoshi Kyoto Univ Sience Assistant Professor, 理学部, 助手 (30183049)
村田 昌之 京都大学, 理学部, 助手 (50212254)
伊藤 忠直 京都大学, 理学部, 助教授 (90093187)
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| Project Period (FY) |
1991 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥30,700,000 (Direct Cost: ¥30,700,000)
Fiscal Year 1993: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1992: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1991: ¥23,800,000 (Direct Cost: ¥23,800,000)
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| Keywords | Time-resolved fluorescence confocal microscope / Endocytosis / Endosome / Lysosome / Sorting mechanism / Low density lipoprotein / Mutant cell / CHO cell / 共焦点蛍光顕微鏡 / エンドソーム / 膜融合 / 時間分解顕微蛍光法 / 共焦点レーザー顕微鏡 / 蛍光共鳴エネルギー移動 / エンドサイト-シス / エンドソ-ム / 共焦点レ-ザ-顕微鏡 / 蛍光共鳴エネルギ-移動 |
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| Research Abstract |
To investigate the process of endocytosis in a living cell in real time, we have developed a new microscopic system, in which a time-resolved fluorescence apparatus is combined with a fluorescence confocal microscope. The time resolved fluorescence apparatus is composed of a pulse laser (wave length : 365 nm ; pules width : 15 picosecond ; output power : several tens pJ/pulse) and a synchroscan streak camera which can record decay time of the pulse laserexcited fluorescence emission in real time, and the confocal fluorescence microscope has a spatial resolution of >1mum. Using system, it is possible to investigate the dynamic process of endocytosis of fluorescent ligand in a living cell in real time with a spatial resolution of > 1mum.
We have developed a novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. RET-LDL particles is endocytosed by cells and relief of the RET upon the disintegration of the LDL in lysosme is monitored by flow cytometory. This method enables us to monitor the process of endocytosis from endosome to lysosome without disruption of the cells. Using this method, we firstly demonstrated that protein phospholylation plays essential role in the transport of LDL particles from endosome to lysosome in CHO cells.
We isolated a mutant cell line of CHO cells which is deficient in the transport of endocytosed ligand from endosome to lysosome, using the abovementioned method. We analyzed the mutation site by the fluorescence timeresolved confocal microscope and cell fractionation, and showed that the mutation site might be at the point where the pathway of endocytosis overlaps the sorting pathway from trans-Golgi network to the lysosome.
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