Molecular Basis of Rod and Cone Photoreceptor Outer Segment Structures

项目摘要/摘要 该项目研究控制脊椎动物光感受器外部的分子和细胞机制。 片段(OS)结构是光感受器细胞生物学中尚未解决的基本问题。这门基础科学 知识差距严重限制了临床对人类致盲疾病的了解(和治疗)，以及 由破坏OS结构的突变引起的动物。各种各样的遗传性视网膜变性 (IRD)是由破坏OS细胞器结构的突变引起的。因为需要正常的操作系统结构 对于健康的视力但尚不清楚，至关重要的是要提前了解数百名 每个杆体和锥体所需的膜盘都具有适当的形状和稳定性。 由异常操作系统结构触发的最古老和最知名的IRD示例是由 外周蛋白-2/rds(P/rds)突变。这个建议建立在我们在上一个项目中演示的基础上 P/RDS直接产生膜曲率的周期，精确雕刻数百个膜盘 圆柱体和圆锥体OSS中存在轮缘。这一进展为将军们提供了一个明确的机制解释 P/rds突变对OS结构的影响，但留下了P/rds如何发生的关键和根本问题 活动是受调控的，以及P/RD的遗传缺陷如何产生多样化的疾病表型。我们的中央 假说认为P/rds的功能是通过与Rom1和富含谷氨酸的异型相互作用来调节的 蛋白质(GARP)是导致视杆细胞和视锥细胞视盘结构不同的原因。研究策略 因此，我将确定P/RD如何与ROM1和GARP合作来构建和维护正常操作系统 结构，并将研究这些蛋白质对杆状和锥状圆盘结构的意义以及如何 突变可以破坏细胞器结构，引发特定的视网膜疾病。 一种利用人类分子遗传学、体外生物化学在纤维素中的贡献的综合方法 将利用工程脊椎动物模型的功能分析和原位分析来进行 研究。具体目标1将阐明P/RD的异型(ROM1和GARP)相互作用的意义 OS磁盘架构的结构、分子功能和支持。具体目标2将确定差异化 P/RDS对杆状和圆锥体结构和生存能力的贡献。 总体而言，这些研究有望阐明正常的杆状和锥状OS磁盘结构是如何产生的，以及 由光感受器特有的蛋白质稳定，这种蛋白质对人类视网膜健康和视力至关重要。这部作品 也将提供一个改进的机制基础，以了解P/RD的致病突变如何 以不同的方式撞击杆和锥体，以产生广泛的异质性IRD。

Project Summary / Abstract This project investigates the molecular and cellular mechanisms that govern vertebrate photoreceptor outer segment (OS) structure, a fundamental unsolved problem in photoreceptor cell biology. This basic science knowledge gap severely limits clinical understanding of (and treatments for) blinding diseases in humans and animals caused by mutations that disrupt OS structure. A broad variety of inherited retinal degenerations (IRDs) are caused by mutations that disrupt OS organelle structure. Because normal OS structure is required for healthy vision but is not yet understood, it is essential to advance knowledge of how the many hundreds of membranous disks required by each rod and cone are properly shaped and stabilized. The most longstanding and well-known examples of IRDs triggered by abnormal OS structure are caused by mutations in peripherin-2/rds (P/rds). This proposal builds upon our demonstration in the previous project period that P/rds directly generates membrane curvature, to precisely sculpt the hundreds of membranous disk rims present in rod and cone OSs. This advance provides a clear mechanistic explanation for the general effects of P/rds mutations on OS structure, but leaves open the critical and fundamental questions of how P/rds activity is regulated, and how inherited defects in P/rds generate a diversity of disease phenotypes. Our central hypothesis is that regulation of P/rds function by heterotypic interactions with rom1 and glutamic acid-rich proteins (GARPs) contributes to differences between rod and cone OS disk structures. The research strategy will therefore identify how P/rds works together with rom1 and GARPs to build and maintain normal OS structure, and will investigate the significance of these proteins for rod vs. cone disk architectures and how mutations can disrupt organelle structure to trigger particular retinal diseases. An integrated approach using contributions from human molecular genetics, in vitro biochemistry, in cellulo functional assays, and in situ analyses of engineered vertebrate animal models will be utilized to carry out the research. Specific Aim 1 will elucidate the significance of heterotypic (rom1 and GARP) interactions for P/rds structure, molecular function, and support of OS disk architecture. Specific Aim 2 will identify differential contributions of P/rds to rod and cone structures and viability. Overall, these studies are expected to clarify how normal rod and cone OS disk structures are generated and stabilized by photoreceptor-specific proteins that are essential for human retinal health and vision. This work will also provide an improved mechanistic basis for understanding how pathogenic mutations in P/rds can differentially impact rods and cones to produce a broad heterogeneity of IRDs.