Molecular Basis of Protein Transport in Photoreceptor

项目摘要/摘要 杆状外段(OS)是一种改良的纤毛，含有约1,000层堆叠的盘膜，这些膜是 浓密地堆积着视觉色素视紫红质。哺乳动物的OS每10天更新一次；新的 光盘是通过一种鲜为人知的机制在操作系统的底部组装的。内部部分， 生物合成和内吞途径所在的位置通过轴丝与OS相连 连接纤毛。视紫红质从合成部位到视盘细胞膜的精确路径 而椎间盘再生的机制还没有完全阐明。然而，这些问题 不仅具有生物学意义，而且还具有临床意义，因为大量的视网膜 退行性疾病表现为视紫红质定位错误和椎间盘组织紊乱。我们的 先前的研究表明，在哺乳动物的视杆中，新的视盘是通过 Smad受体激活锚(SARA)介导的轴膜视紫红质小泡之间的融合 和新生的光盘。视紫红质囊泡为视盘提供“积木”的新概念 膜提出了许多问题。小泡的性质是什么？什么是分子和 这些囊泡的产生和传递背后的调控途径？因为莎拉是 一种直接与视紫红质结合的早期/分选内体蛋白及其生物合成途径 大多数顶端表面蛋白都与内吞途径相互作用，我们将检验这一假说。 高尔基体视紫红质在到达OS(特异性Aim1)的过程中穿过胞内隔室。多么 视紫红质的靶向性受到干扰内皮细胞转运过程或 将检查特定的内体隔室的功能。此外，我们将测试 假说Sara除了在调节视紫红质的囊泡运输方面也有作用。 在椎间融合中的作用(特定目标2)。一些可缺失SarA基因的小鼠模型 在这些研究中，将采用组织特异性和/或时间调控方式下的杆状细胞。 最后，我们将检验我们的假设，即环境照明在调节 OS的传递，因此，视紫红质的盘结合(特异性Aim3)。转基因的啮齿动物杆子， 条件基因敲除小鼠、细胞培养模型和几种创新技术将被用于 全面排查这些问题。

PROJECT SUMMARY/ABSTRACT The rod outer segment (OS) is a modified cilium containing ~1,000 stacked disc membranes that are densely packed with the visual pigment rhodopsin. The mammalian OS is renewed every 10 days; new discs are assembled at the bases of the OS by a poorly understood mechanism. The inner segment, where the biosynthetic and the endocytic pathways are housed, is linked with the OS via the axonemal connecting cilium. The precise route taken by rhodopsin from its site of synthesis to the disc membrane and the mechanism underlying disc renewal are not completely elucidated. However, these questions are not only biologically interesting, but are also clinically relevant because a large number of retinal degenerative diseases are manifested by rhodopsin mislocalization and disc disorganization. Our previous studies suggested that in mammalian rods the new discs are assembled and "grown" via Smad anchor for receptor activation (SARA)-mediated fusions between axonemal rhodopsin vesicles and nascent discs. The new concept that rhodopsin vesicles provide "building blocks" for disc membranes raises many questions. What is the nature of the vesicles? What are the molecular and regulatory pathways that underlie the production and the delivery of these vesicles? Because SARA is an early/sorting endosomal protein that directly binds to rhodopsin and the biosynthetic pathways of most apical surface proteins interact with the endocytic pathway, we will test the hypothesis that post- Golgi rhodopsins traverse the endocytic compartments en route to the OS (Specific Aim1). How rhodopsin's targeting is affected by strategies interfering with either the endocytic trafficking process or the functions of specific endosomal compartments will be examined. Furthermore, We will test the hypothesis that SARA also has a role in regulating the vesicular trafficking of rhodopsin in addition to its role in the disc fusion (Specific Aim 2). A number of mouse models in which sara gene can be deleted in rods under tissue-specific and/or temporally-regulated fashion will be employed in these studies. Finally, we will test our hypothesis that environmental lighting plays an important role in regulating the OS delivery, and, hence, disc incorporation of rhodopsin (Specific Aim3). Transfected rodent rods, conditional knockout mice, cell culture models, and several innovative techniques will be employed to comprehensively investigate these questions.