Molecular Basis of Protein Transport in Photoreceptor

描述（由申请人提供）：视杆细胞在形态和功能上高度极化和区室化。许多影响视网膜的遗传性疾病表现为视色素视紫红质的运输和视杆细胞外段（ROS）的形态发生和/或更新。我们以前的研究表明，视紫红质的C-末端是视网膜色素变性（RP）相关突变的热点，它包含其ROS传递的寻址代码。我们还表明，细胞质动力蛋白为基础的运动是必不可少的视紫红质内节的定向运输。在这个应用程序中，我们建议调查的机械和机制，移动视紫红质在其他不同的细胞领域的杆和他们参与ROS的形成和维护。在目标1中，我们提出了识别捕获，保留，导航和/或融合到顶端表面上的视紫红质装载囊泡的分子。本研究将使用极化MDCK上皮细胞模型，这已被证明是有用的研究视紫红质的矢量运输。目的2研究视紫红质与一种新的视紫红质C端相互作用蛋白SARA的相互作用的生理相关性。已经显示FYVE结构域特异性结合磷脂酰肌醇-3 '-磷酸，并且SARA参与蛋白质运输和膜融合。我们的超微结构分析揭示了一个独特的分布在ROS轴丝近端部分的新生盘膜附近的囊泡/小管上的SARA。我们计划通过采用转染的啮齿动物视网膜作为模型系统来测试体内SARA的扰动如何影响视紫红质靶向和椎间盘形态发生。相反，也将检查用不能结合SARA的RP突变视紫红质转染的视杆细胞的表型。在目标3中，我们将确定参与活性氧中SARA介导的膜融合途径的细胞组分，并测试它们在体内视紫红质靶向和椎间盘生物发生中的重要性。成功地实现所提出的具体目标将显着进一步深入了解的分子基础的起源和维持的极性的视觉细胞。这些主题是细胞生物学和视觉研究的核心兴趣。最后，这些研究对于我们理解各种退行性视网膜疾病的病因是高度相关的，并且对于未来患病视网膜的合理治疗具有重要意义。

DESCRIPTION (provided by applicant): The rod cell is highly polarized and compartmentalized in both morphology and function. A number of genetic disorders affecting the retina are manifested in the trafficking of the visual pigment rhodopsin and the morphogenesis and/or renewal of the rod outer segment (ROS). Our previous studies showed that rhodopsin's C-terminus is a hot spot for mutations associated with retinitis pigmentosa (RP) and it contains the addressing code for its ROS delivery. We also showed that cytoplasmic dynein-based motility is essential for the directional transport of rhodopsin within the inner segment of rod. In this application, we propose to investigate the machinery and mechanisms that move rhodopsin within other distinct cellular domains of rod and their involvement in ROS formation and maintenance. In Aim 1, we propose to identify molecules that capture, retain, navigate, and/or fuse rhodopsin-laden vesicles onto apical surfaces. This study will use the model of polarized MDCK epithelial cells, which have proven useful for studying the vectorial transport of rhodopsin. Aim 2 will study the physiological relevance of the interaction between rhodopsin and the FYVE domain-containing protein SARA, a novel rhodopsin C-terminus interacting protein. It has been shown that the FYVE domain specifically binds phosphatidylinositiol-3'-phosphate, and SARA is involved in protein trafficking and membrane fusion. Our ultrastructural analysis reveals a unique distribution of SARA on the vesicles/tubules near nascent disc membranes in the proximal portion of ROS axoneme. We plan to test how the perturbation of SARA in vivo might affect rhodopsin targeting and disc morphogenesis by employing transfected rodent retinas as a model system. Conversely, the phenotypes of rods transfected with RP mutant rhodopsins that fail to bind to SARA will also be examined. In Aim 3, we will identify the cellular components involved in the SARA-mediated membrane fusion pathway in the ROS and test their importance in rhodopsin targeting and disc biogenesis in vivo. Successful achievement of the proposed specific aims will significantly further our insights into the molecular basis of the genesis and maintenance of the polarity of visual cells. These topics are a central interest in cell biology and vision research. Finally, these studies are highly relevant for our understanding of the etiology of various degenerative retinal diseases and have important implications for future rational therapies for the diseased retina.