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

本研究的主要目的是用单分子成像技术阐明脊椎动物光感受器盘膜中G蛋白转导蛋白的作用机制。第二个目的是研究光适应或暗适应过程中G蛋白在视杆感受器内外节间转运的分子机制。作为第一个要完成的任务，PI挑战了用近红外荧光化合物(Hilyte750)标记转导蛋白，并成功了。标记的转导蛋白在结合光漂白的视紫红质、核苷酸交换活性和PDE6激活方面保持其正常活性。利用这种荧光标记的转导蛋白，在750 nm的激光激发下，PL成功地观察到了暗状态下转导蛋白的单分子运动。转导蛋白在暗适应的视盘细胞膜中表现出比视紫红质更强的Faser扩散。在可见光下，转导蛋白与光漂白的视紫红质形成复合体，该复合体表现出缓慢的…比视紫红质更易扩散。转导蛋白-视紫红质复合体的扩散速率依赖于膜中胆固醇的含量，即胆固醇结合化合物甲基-β-环糊精对胆固醇的清除加速了转导蛋白-视紫红质复合体的扩散，提示转导蛋白的激活发生在视紫红质膜胆固醇依赖微区的外围。虽然微区形成的机制尚未阐明，但这些结果使我们对脊椎动物光传导系统中G蛋白的信号机制有了更深层次的新认识。至于转导蛋白在视杆感受器内外节间的转运，Pl尚不能将荧光标记的转导蛋白引入视杆细胞外段。然而，Pl发现GTP形式的转导蛋白倾向于在周围的斑点形成聚集，似乎是盘膜切口处的微管蛋白纤维，这表明转导蛋白与微管蛋白之间的相互作用在运输过程中的重要性。有关转导蛋白及其与视紫红质的复合体的单分子行为的论文目前正在准备中。较少

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