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细丝蛋白A分子的力学性质与肌动蛋白/细丝蛋白A凝胶之间的关系。1.利用荧光显微镜研究微丝蛋白A在肌动蛋白/微丝蛋白A凝胶中的去折叠和重折叠。以荧光染料俄勒冈州绿色标记hsFLNa。在hsFLNa的天然状态下，由于自猝灭，荧光强度较低，并且在存在5 M盐酸胍的情况下，由于hsFLNa的去折叠减少了自猝灭，荧光强度增加到天然状态下的4.3倍。2.在含100 mM KCl的F-缓冲液中，成功地制备了肌动蛋白/细丝蛋白A巨脂质体凝胶。我们可以在低肌动蛋白浓度（2 μM）下使用德克萨斯红-X鬼笔环肽在凝胶中观察到荧光标记的F-肌动蛋白。我们发现，低浓度（<cmc）的溶血磷脂酰胆碱（lyso-PC）诱导的囊泡分裂的GUV的液体有序（lo）相膜。在肌动蛋白/细丝蛋白A的凝胶中，我们没有观察到F-actin的起伏运动和蠕动，而在没有细丝蛋白A的情况下，F-actin的起伏运动和蠕动被抑制了。为了验证原子力显微镜测量生物素与亲和素之间解结合力的方法，我们尝试用生物素共价连接的原子力显微镜探针测量生物素与亲和素之间的解结合力，并成功地得到了与其他方法相似的解结合力值。

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.