| Project/Area Number |
11480210
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | Osaka University |
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Principal Investigator |
SAKO Yasushi Graduate School of Medicine, Osaka University, Associate Professor, 医学系研究科, 助教授 (20215700)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,200,000 (Direct Cost: ¥12,200,000)
Fiscal Year 2001: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1999: ¥6,600,000 (Direct Cost: ¥6,600,000)
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| Keywords | total internal reflection / fluorescence / EGF / Ras / signal transaction / FRET / リン酸化 / 成長因子 / 蛍光励起エネルギー移動 / 1分子測定 / 情報伝達 / EGF / Ras / Raf / GFP / 励起エネルギー移動 |
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| Research Abstract |
The aim of this study was the development of new techniques to visualize single molecules of proteins in living cells to observe protein-protein interactions. The techniques were applied to the studies of the reactions of cell signaling molecules. Visualization of single molecules was achieved by using total internal reflection fluorescence microscopy. Single molecules of epidermal growth factor (EGF), EGF receptor (EGFR) and related signaling molecules were detected.
During the period of this grant, we obtained the following achievements.
(1) Visualization of cell signaling molecules: EGFR, small G-protein Ras and cytoplasmic serine/threonine kinase c-Raf1 were tagged with green fluorescent protein (GFP) or its derivatives without affecting their biological activities and were visualized as single molecules in living cells.
(2) Detection of molecular interactions between EGF/EGFR complexes: a mixture of Cy3-EGF and Cy5-EGF was added to cells expressing EGFR and fluorescence resonance ene
… More
rgy transfer (FRET) from single Cy3-EGF/EGFR complex to another single Cy5-EGF/EGFR complex was detected on a living cell surface. Fluctuation of the FRET efficiency was observed suggesting structural fluctuation of EGFR dimers.
(3) Pre-clustering of EGFRs: an EGFR-null cell (CHO-K1) was transfected with EGFR-GFP and variation of the fluorescence intensity of EGFR-GFP spot on the cell surface was assessed. The result indicated that EGFRs formed pre-clusters before addition of EGF. Artificial modification of the cluster size distribution affected tyrosine phosphorylation induced by EGF-binding.
(4) Molecular interactions between Ras and Raf1: in cells transfected with both Ras and Raf1, stimulation with EGF induced translocation of Raf1 from the cytoplasm to the plasma membrane. Direct molecular interactions of Ras and Raf1 were visualized by FRET imaging from GFP-Raf1 to YFP-Ras. The interactions were prolonged at the membrane raffles for more than 60 min. Both Raps and Raf1 visualized in single molecules were rapidly diffusing even in the membrane raffles indicating the concentration of active molecules in the raffles was maintained by dynamic equilibrium. Less
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