Single Molecular Biochemistry of The Operating Mechanism of Photoreoeptor G protein and its Trafficking Mechanism.

光感受器G蛋白运作机制及其运输机制的单分子生物化学。

• 批准号: 18310088
• 负责人: HAYASHI Fumio
• 金额: $ 9.46万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18310088/
• 关键词: single molecule observation; transducin; G protein; rhodopsin; phototransduction; hotorecentor; trafficking; disk membrane; G蛋白質; トラフィックング

The main purpose of this research is to clarify the operating mechanism of G protein transducin in vertebrate photoreceptor disk membranes by single molecular imaging technique. The second purpose is to examine the molecular mechanism of G protein trafficking between inner and outer segment of rod photoreceptor during light- or dark adaptation. As the first task to clear, PI challenged to label transducin with near-infrared fluorescent compound (Hilyte 750), and succeeded. The labeled transducin retained its normal activity in binding light-bleached rhodopsin, nucleotide exchange activity, and PDE6 activation. By using this fluorescent-labeled transducin, Pl succeeded to observe single molecular motion of transducin in the dark state by using 750 nm laser beam for excitation of the label. Transducin showed much faser diffusion than rhodopsin in dark-adapted disk membrane. Upon exposure to visible light, transducin forms complex with light-bleached rhodopsin, and the complex showed slow … More er diffusion than rhodopsin. The diffusion rate of the complex was dependent on cholesterol-content of the disk membrane, i.e. the cholesterol-depletion by cholesterol-binding compound methyl-beta-cyclodextrin speeded up the diffusion of transducin-rhodopsin complex, suggesting that the activation of transducin occurs at the peripheries of cholesterol-dependent microdomains in the disk membrane. Although the mechanism of the microdomain formation has not been elucidated, these result gave us a deeper and new insight I into the G protein signaling mechanism of vertebrate phototransduction system. As for the trafficking of transducin between inner and outer segment of rod photoreceptor, Pl has not been able to introduce fluorescent-labeled transducin into rod outer segment. However, Pl's finding that GTP-form transducin shows propensity to form aggregation at the peripheral spots seemingly tubulin fibers at the mouth of incisures of the disk membrane suggested the importance of the interaction between transducin with tubulin in the trafficking. Papers about the single molecular behavior of transducin and its complex with rhodopsin are now in preparation. Less

本研究的主要目的是通过单分子成像技术阐明脊椎动物感光盘膜中G蛋白转导蛋白的运行机制。第二个目的是研究光适应或暗适应过程中视杆光感受器内节和外节之间 G 蛋白运输的分子机制。作为第一个要解决的任务，PI挑战用近红外荧光化合物（Hilyte 750）标记转导蛋白，并成功了。标记的转导蛋白在结合光漂白视紫红质、核苷酸交换活性和 PDE6 激活方面保留了其正常活性。通过使用这种荧光标记的转导蛋白，Pl通过使用750 nm激光束激发标记，成功地观察了暗状态下转导蛋白的单分子运动。在暗适应盘膜中，转导蛋白比视紫红质表现出更快的扩散。暴露于可见光后，转导蛋白与光漂白的视紫红质形成复合物，并且该复合物表现出比视紫红质慢的扩散。该复合物的扩散速率取决于椎间盘膜的胆固醇含量，即胆固醇结合化合物甲基-β-环糊精的胆固醇消耗加速了转导蛋白-视紫红质复合物的扩散，表明转导蛋白的激活发生在椎间盘中胆固醇依赖性微结构域的外围 膜。尽管微结构域形成的机制尚未阐明，但这些结果使我们对脊椎动物光转导系统的G蛋白信号传导机制有了更深入、新的认识。至于转导蛋白在视杆光感受器内节和外节之间的运输，P1未能将荧光标记的转导蛋白引入视杆外节。然而，Pl发现GTP型转导蛋白显示出在盘膜切口口处看似微管蛋白纤维的外围点处形成聚集的倾向，这表明转导蛋白与微管蛋白之间的相互作用在运输中的重要性。关于转导蛋白及其与视紫红质复合物的单分子行为的论文正在准备中。较少的

项目成果

カエル視細胞のロドプシンは二量体化によって脂質ラフトへ移行する

青蛙感光细胞中的视紫红质通过二聚化迁移至脂筏

• 发表时间: 2007
• 作者: 妹尾圭司;林 文夫
• 通讯作者: 林 文夫

Single Molecule Tracking of Transducin and Rhodopsin in Rod Photoreceptor Disk Membranes

杆感光盘膜中转导蛋白和视紫红质的单分子追踪

• 发表时间: 2007
• 作者: Fumio;Hayashi;Shibata;Nobuyuki;Toshi;Takahashi;Keiji;Seno
• 通讯作者: Seno

視細胞円板膜上でのトランスデューシン及びロドプシンの一分子追跡

感光盘膜上转导蛋白和视紫红质的单分子追踪

• 发表时间: 2007
• 作者: 林 文夫;他4名
• 通讯作者: 他4名

Frog photoreceptor rhodopsin is recruited to the raft upon dimerization

青蛙光感受器视紫红质在二聚化后被募集到筏上

• 发表时间: 2007
• 作者: Keiji;Seno;Fumio;Hayashi
• 通讯作者: Hayashi