Molecular Basis of Protein Transport in Photoreceptor

描述(申请人提供)：杆状细胞高度极化，在形态和功能上都是分开的。许多影响视网膜的遗传性疾病表现为视觉色素视紫红质的运输和视杆外节(ROS)的形态形成和/或更新。我们以前的研究表明，视紫红质的C末端是与视网膜色素变性(RP)相关的突变热点，它包含ROS传递的地址代码。我们还发现，基于胞浆动力蛋白的运动对于视紫红质在视杆内段的定向运输是必不可少的。在这一应用中，我们建议研究视紫红质在视杆细胞其他不同区域内移动的机制和机制，以及它们参与ROS的形成和维持。在目标1中，我们建议识别捕获、保留、导航和/或融合含有视紫红质的囊泡到心尖表面的分子。这项研究将使用极化的MDCK上皮细胞模型，该模型已被证明对研究视紫红质的载体运输是有用的。目的2研究视紫红质与含有FYVE结构域的蛋白SARA之间相互作用的生理学相关性。研究表明，FYVE结构域与磷脂酰肌醇-3‘-磷酸特异性结合，SARA参与蛋白质转运和膜融合。我们的超微结构分析显示，在ROS轴丝近端的新生盘膜附近的小泡/小管上有一种独特的SARA分布。我们计划通过使用转基因的啮齿动物视网膜作为模型系统来测试体内SARA的扰动可能如何影响视紫红质靶向和视盘形态发生。反之，也将检测不能与SARA结合的RP突变型视紫红质转基因棒的表型。在目标3中，我们将在ROS中鉴定参与SARA介导的膜融合途径的细胞成分，并测试它们在体内视紫红质靶向和视盘生物发生中的重要性。所提出的特定目标的成功实现将极大地促进我们对视觉细胞极性发生和维持的分子基础的洞察。这些主题是细胞生物学和视觉研究的中心兴趣。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具有很高的相关性，并对未来视网膜疾病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.