Cilia Assembly and Transport in the Vertebrate Retina

描述（由申请人提供）：光感受器的发育和存活需要蛋白质从内段到外段的有效运输。该提案的中心假设认为，鞭毛内运输（IFT）蛋白对于外节段的形成和通过连接纤毛的蛋白质运输至关重要，而IFT基因的突变导致视网膜变性。IFT蛋白形成一个多亚基复合物，通过运动蛋白马达的作用沿着连接纤毛的微管运输。我们将使用斑马鱼作为实验系统，因为斑马鱼的光感受器具有很好的特征，在斑马鱼中存在成熟的技术来操纵基因表达并创造遗传马赛克胚胎，我们有三个IFT基因的斑马鱼突变体。这些突变体表现为视紫红质定位错误、光感受器外段形成中断和光感受器变性。我们的目标是了解IFT颗粒在光感受器内纤毛蛋白运输中的功能和调控。具体而言，我们建议：1)通过电镜和光镜结合免疫组织化学检测三种IFT蛋白的外段结构和蛋白定位，确定其突变对斑马鱼光感受器发育的功能和影响。2)研究Par-aPKC极性复合物对IFT颗粒组装和功能的调控。IFT粒子和Par-aPKC复合物在纤毛内共定位，并与激酶- 1相互作用。我们将使用免疫沉淀和遗传镶嵌分析来研究这些复合物之间的相互作用。3)利用荧光显微镜和延时共聚焦显微镜分析IFT颗粒中组装的IFT- gfp融合蛋白的定位和运动，测量IFT57突变体中IFT复合物的稳定性和运动。4)利用morpholino寡核苷酸生成斑马鱼Bardet-Biedl综合征表型，确定各IFT蛋白在光感受器形态发生中的需求，并通过光镜和荧光显微镜评估光感受器的发育。由于光感受器发育所需的蛋白质转运机制也在成人光感受器的维持中起作用，我们的研究可以为导致视网膜病变的过程提供见解。

DESCRIPTION (provided by applicant): Photoreceptor development and survival requires the efficient transport of proteins from the inner segment to the outer segment. The central hypothesis of this proposal states that intraflagellar transport (IFT) proteins are essential for outer segment formation and protein transport through the connecting cilium and mutations in IFT genes cause retinal degeneration. The IFT proteins form a multisubunit complex that is transported along microtubules in the connecting cilium via the action of kinesin motors. We will use zebrafish as an experimental system because zebrafish photoreceptors are well-characterized, established techniques exist in zebrafish to manipulate gene expression and create genetic mosaic embryos, and we have zebrafish mutants for three IFT genes. These mutants exhibit mislocalized rhodopsin, disrupted photoreceptor outer segment formation, and photoreceptor degeneration. Our objective is to understand the function and regulation of the IFT particle in ciliary protein transport within the photoreceptor. Specifically, we propose to: 1) To determine the function and effects of mutations of three IFT proteins on zebrafish photoreceptor development by examining outer segment structure and protein localization using electron microscopy and light microscopy combined with immunohistochemistry. 2) To examine the regulation of IFT particle assembly and function by the Par-aPKC polarity complex. The IFT particle and Par-aPKC complex co-localize within the cilium and both interact with kinesin-ll. We will investigate the interactions between these complexes using immunoprecipitation and genetic mosaic analysis. 3) To measure IFT complex stability and movement in IFT57 mutants by analyzing the localization and movement of IFT-GFP fusion proteins assembled in the IFT particle using fluorescence microscopy and time-lapse confocal microscopy. 4) To determine the requirement for each IFT protein in photoreceptor morphogenesis by using morpholino oligonucleotides to generate Bardet-Biedl Syndrome phenotypes in zebrafish and assess photoreceptor development by light and fluorescence microscopy. As the mechanisms of protein transport required for photoreceptor development also function in the maintenance of adult photoreceptors, our studies can provide insights in the processes that lead to retinal pathologies.