Cilia Assembly and Transport in Photoreceptor Cells

项目描述（申请人提供）：该项目的长期目标是了解脊椎动物光感受器细胞中纤毛形成和维持的分子基础。在脊椎动物中，光感受器外节的组装和维护始于连接纤毛的形成。连接的纤毛包含一个以微管为基础的轴突，该轴突被一个基底固定在根尖的内段上。纤毛的形成始于内节顶端的基部对接。基因突变破坏基底体和/或纤毛的组装、结构或功能，导致被称为纤毛病的一系列疾病。这些多综合征性疾病通常表现为视网膜变性、肾脏疾病、智力迟钝和多指畸形。在当前的应用中，我们将利用斑马鱼的功能丧失策略来研究控制基底体定位的机制。在Specific Aim 1中，我们将通过检测动力蛋白和动力蛋白p150和p50亚基的斑马鱼突变体，来验证动力蛋白/动力蛋白复合物在纤毛形成之前调节基底体的顶端运输的假设。在具体目标2中，我们提供了初步证据，表明基底体在成年斑马鱼视网膜内显示出高度极化的排列。我们将直接测试PCP通路调节这种模式并对光感受器存活至关重要的假设。在特异性目标3中，我们将检查携带Joubert综合征基因arl13b零突变的斑马鱼的视网膜表型。提出的实验还将测试GTPase结构域和纤毛靶向序列RVxPx对Arl13b功能的要求。我们还将测试arl13b与Bardet-Biedl综合征（BBS）基因和PCP通路组分的功能相互作用。这些相互作用将确定潜在的第二位点修饰剂，增强光感受器表型的表达。这些研究的结果将揭示在纤毛形成之前基底体放置所需的新机制，并确定影响遗传性失明的新的遗传相互作用。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular basis of cilia formation and maintenance in vertebrate photoreceptor cells. In vertebrates, the assembly and maintenance of photoreceptor outer segments begins with the formation of a connecting cilium. The connecting cilium contains a microtubule- based axoneme that is anchored to the apical inner segment by a basal body. Cilia formation begins with the docking of basal bodies at the apical surface of the inner segment. Genetic mutations disrupting the assembly, structure, or function of basal bodies and/or cilia result in a spectrum of diseases known as ciliopathies. These multisyndromic disorders often present with retinal degeneration, kidney disease, mental retardation, and polydactyly. In the current application, we will utilize loss-of-function strategies in zebrafish to investigate the mechanisms controlling basal body localization. In Specific Aim 1, we will test the hypothesis that the dynein/dynactin complex regulates the apical transport of basal bodies preceding cilia formation by examining zebrafish mutants in dynein and the p150 and p50 subunits of dynactin. In Specific Aim 2, we provide preliminary evidence that basal bodies show a highly polarized arrangement within the adult zebrafish retina. We will directly test the hypothesis that the PCP pathway regulates this patterning and is essential for photoreceptor survival. In Specific Aim 3, we will examine zebrafish carrying null mutations in the Joubert Syndrome gene arl13b for retinal phenotypes. Proposed experiments will also test the requirement of the GTPase domain and a ciliary-targeting sequence RVxPx for Arl13b function. We will also test arl13b for functional interactions with Bardet-Biedl Syndrome (BBS) genes, and components of the PCP pathway. These interactions will identify potential second-site modifiers that enhance expression of photoreceptor phenotypes. The results of these studies will reveal novel mechanisms required for basal body placement prior to cilia formation and to identify novel genetic interactions that influence hereditary blindness.