Molecular Basis of Protein Transport in Photoreceptor

项目总结/摘要 杆外节（OS）是一种修饰的纤毛，包含约1，000个堆叠的盘膜， 密集地包裹着视色素视紫红质。哺乳动物操作系统每10天更新一次;新 盘通过不太了解的机制组装在OS的底部。内部部分， 其中生物合成和内吞途径被容纳，通过轴丝与OS连接 连接纤毛视紫红质从其合成部位到视盘膜的精确路线 和椎间盘更新的机制尚未完全阐明。然而，这些问题 不仅在生物学上是有趣的，而且在临床上也是相关的，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紊乱。我们 先前的研究表明，在哺乳动物的视杆细胞中，新的视杆细胞是通过 Smad锚受体激活（SARA）介导的轴丝视紫红质囊泡之间的融合 和新生的椎间盘视紫红质囊泡为椎间盘提供“积木”的新概念 膜引起了许多问题。囊泡的性质是什么？什么是分子和 这些囊泡的产生和传递的调控途径？因为莎拉是 一种直接结合视紫红质的早期/分选内体蛋白， 大多数顶端表面蛋白与内吞途径相互作用，我们将测试后- 高尔基体视紫红质穿过内吞区室到达OS（特异性目的1）。如何 视紫红质的靶向受到干扰内吞运输过程或 将检查特定内体区室的功能。此外，我们将测试 假设SARA也有调节视紫红质的囊泡运输的作用，除了它的 在椎间盘融合中的作用（具体目标2）。sara基因缺失小鼠模型的建立 在这些研究中，将采用组织特异性和/或时间调节方式下的杆中。 最后，我们将测试我们的假设，即环境照明在调节 OS递送，以及因此视紫红质的椎间盘掺入（特异性Aim 3）。转染的啮齿类杆状病毒， 条件性基因敲除小鼠、细胞培养模型和一些创新技术将被用于 全面调查这些问题。

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.