| Project/Area Number |
15510099
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nanomaterials/Nanobioscience
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| Research Institution | Shizuoka University |
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Principal Investigator |
YAMAZAKI Masahito Shizuoka Univ., Fac.Science, Associate Professor, 理学部, 助教授 (70200665)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Tadanao Kyoto Univ., Fac.Science, Associate Professor, 理学研究科, 助教授 (90093187)
OHASHI Kazuyo Chiba Univ., Fac.Science, Professor, 理学部, 教授 (90114248)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2003: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Keywords | actin / filamin A gel / mechanical property of single protein / filamin / unfolding of protein / giant liposome (GUV) / fluorescence microscopy / thermal motion of F-actin / vesicle fission / 巨大リボソーム / 秩序液体相 / 原子間力顕微鏡 / 蛋白質と低分子の結合力 / ゲルの浸透圧応答 / フイラミン / ゲルの粘弾性 |
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| Research Abstract |
The purpose of this research was to elucidate the relationship between the mechanical property of single filamin A molecules and actin/filamin A gel. For this purpose, we developed several methods and got some several fundamental data.
1.To investigate unfolding and refolding of filamin A in the actin/filamin A gel using a fluorescence microscope, hsFLNa was labeled with a fluorescent dye (Oregon green). At native state of hsFLNa, the fluorescence intensity was low due to the self-quenching, and in the presence of 5M guanidine hydrochloride it increased to the 4.3-fold of that at the native state because the unfolding of hsFLNa reduced the self-quenching. On the refolding by the dilution, the fluorescence intensity decreased.
2.We succeeded in the preparation of the actin/filamin A gel in a giant liposome (GUV) in F-buffer containing 100 mM KCl by our new method. We could observe fluorescent-labeled F-actin in the gel using Texas Red-X phalloidin at low actin concentration (2 μM). We found that low concentrations (<cmc) of lysophosphatidylcholine (lyso-PC) induced vesicle fission of GUVs of liquid-ordered (lo) phase membrane. When GUVs contained the gel, no vesicle fission was induced by the addition of lyso-PC.
3.We could not observe any undulation motion and reptation of F-actin in the actin/filamin A gel, although in the absence of filamin A, large undulation and reptation of F-actin were observed.
4.To measure the unbinding force between the actin-binding domain (ABD) of filamin Awith F-actin, we produced ABD of chicken filamin. To check the methodology of the measurement of the unbinding force using atomic force microscopy, we tried to measure the unbinding force between avidin and biotin using the biotin covalently-attached AFM tip the supported lo phase membrane containing the biotin-lipid, and succeeded in getting a similar value of the unbinding force obtained by other method.
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