Mechanical Property of Single Filamin A Molecules and the Mechanism for Function of Actin/Filamin A Gel

本研究的目的是阐明单个丝蛋白A分子的力学性质与肌动蛋白/丝蛋白A凝胶之间的关系。为此，我们开发了几种方法，得到了一些基础数据。为了在荧光显微镜下观察肌动蛋白/丝蛋白A凝胶中丝蛋白A的展开和再折叠，用荧光染料（俄勒冈绿）标记hsFLNa。在hsFLNa的天然状态下，由于自猝灭，荧光强度较低，在5M胍盐酸盐存在下，由于hsFLNa的展开减少了自猝灭，荧光强度增加到天然状态下的4.3倍。稀释后再折叠时，荧光强度降低。在含100 mM KCl的f -缓冲液中，我们成功地制备了肌动蛋白/丝蛋白A凝胶。在低肌动蛋白浓度（2 μM）下，用Texas Red-X phalloidin观察凝胶中荧光标记的F-actin。我们发现低浓度（<cmc）溶血磷脂酰胆碱（lyso-PC）诱导液体有序（低）相膜guv的囊泡裂变。当guv中含有该凝胶时，添加lyso- pc不会引起囊泡裂变。在肌动蛋白/丝蛋白A凝胶中，我们没有观察到f -肌动蛋白的波动运动和重复，而在没有丝蛋白A的情况下，我们观察到f -肌动蛋白的大波动和重复。为了测定鸡丝蛋白a的肌动蛋白结合域（ABD）与f -肌动蛋白之间的解结合力，我们制备了鸡丝蛋白的ABD。为了验证原子力显微镜测量生物素与生物素之间解结合力的方法，我们尝试用生物素共价附着的AFM尖端测量生物素与生物素之间的解结合力，并成功地获得了其他方法获得的类似解结合力值。

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.