Structural dissection of the visual signaling network in cone cells

描述（由申请人提供）：脊椎动物视网膜有两种类型的感光细胞-视杆细胞和视锥细胞。视杆细胞对光非常敏感，但视锥细胞对白天的视力、敏锐度和颜色辨别力更重要。视锥细胞可能受到基因突变的不利影响，并可能成为视杆细胞变性的继发性损伤。环境因素和衰老也会影响视锥细胞的存活，从而导致年龄相关性黄斑变性。然而，由于视锥细胞数量少，缺乏合适的研究方法，对视锥细胞的精细结构知之甚少，阻碍了对视锥细胞精细结构的研究。我们这项研究的长期目标是阐明锥细胞光感受器的功能和生存的关键的详细结构特征。视锥细胞的光转导是由视锥色素（视蛋白），膜结合蛋白，原型G蛋白偶联受体（GPCR）的激活。视紫红质占视杆细胞膜中蛋白质的90%，视锥细胞中视蛋白的组成和结构尚未确定。这些信息将广泛适用于其他信号转导级联，因为GPCR代表了已知最大的一类药物，激素和神经肽受体。在这里，我们提出了三个主题相关的具体目标相关的视锥细胞的结构生物学：（1）确定在转基因小鼠缺乏转录因子NRL，增强的S-锥综合征在人类的啮齿动物模型的视锥细胞的详细结构。这些小鼠专门产生锥状光感受器，将通过冷冻电子断层扫描检查。这种转基因物种中丰富的锥体样细胞将允许创建有效提取和成像研究所需的方案。(2)使用冷冻电子断层扫描来阐明尼罗鼠视锥细胞的精细结构，尼罗鼠的视锥细胞约占33%，而大多数其他鼠种的视锥细胞约占1%。这项研究将使我们能够辨别天然锥结构相比，杆在同一物种。此外，在这种昼夜啮齿动物的实验结果应该更普遍的人类感光细胞的超微结构和功能。(3)确定NRL转基因小鼠和尼罗大鼠个体椎间盘膜中锥状色素的组织。这将使我们能够直接观察视杆细胞和视锥细胞之间视色素的组织是否以及如何不同。关于它们的组织的信息将提高对人类疾病状态的理解，其中杆和锥光感受器功能受损。公共卫生相关性：脊椎动物视网膜有两种类型的感光细胞--视杆细胞和视锥细胞。视锥细胞对我们白天的视力、敏锐度和颜色辨别力至关重要，它们的健康受到基因突变、环境因素和衰老的影响。然而，我们对视锥细胞的了解很少，因为它们只占人类视网膜感光细胞的5%。因此，我们研究的长期目标是阐明视锥光感受器的详细结构特征，这些结构特征对它们的功能和生存至关重要。

DESCRIPTION (provided by applicant): Vertebrate retinas have two types of photoreceptor cells - rods and cones. Rods are exquisitely sensitive to light but cones are more critical for daytime vision, acuity and color discrimination. Cone cells can be adversely affected by genetic mutations and can become secondary casualties of rod degeneration. Environmental factors and aging also can affect cone survival, thereby contributing to age-related macular degeneration. However, little is known about the fine structure of cones due to the paucity of cone cells and lack of proper methods to investigate them has impeded studies of the fine structure. Our long-term objective of this research is to elucidate the detailed structural features of cone photoreceptors critical for their function and survival. Cone phototransduction is initiated by activation of cone pigments (opsins), membrane-bound proteins that are prototypic G protein-coupled receptors (GPCRs). While rhodopsin comprises ~90% of protein in rod disc membranes, the composition and organization of opsins in cone cells have yet to be determined. Such information would be broadly applicable to other signal transduction cascades because GPCRs represent the largest known class of drug, hormone and neuropeptide receptors. Here we propose three thematically linked specific aims related to the structural biology of cone cells: (1) Determine the detailed structure of cone cells in transgenic mice lacking transcriptional factor NRL, a rodent model of enhanced S-cone syndrome in humans. These mice exclusively produce cone-like photoreceptors that will be examined by cryo-electron tomography. The abundance of cone-like cells in this transgenic species will allow creation of protocols required for efficient extraction and imaging studies. (2) Use cryo-electron tomography to elucidate the fine structure of cones in the Nile rat, an animal that has ~33% cone cells compared to ~1% in most other rat species. This study will allow us to discern the native cone structure as compared with rods in the same species. Moreover, the experimental findings in this diurnal rodent should be more generalizable to human photoreceptor ultrastructure and function. (3) Determine the organization of cone pigments in individual disc membranes from both NRL transgenic mice and Nile rats. This will allow us to observe directly if and how the organization of visual pigments differs between rod and cone cells. Information about their organization would improve understanding of human disease states in which rod and cone photoreceptor function is impaired. PUBLIC HEALTH RELEVANCE: Vertebrate retinas have two types of photoreceptor cells - rods and cones. Cones are critical for our daytime vision, acuity and color discrimination and their health is affected by genetic mutations, environmental factors and aging. Yet our knowledge of cones is scanty because they comprise only about 5% of photoreceptor cells in the human retina. Thus, the long-term objective of our research is to elucidate the detailed structural features of cone photoreceptors central to their function and survival.