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节到外节的有效运输。该提议的中心假设指出，鞭毛内转运（IFT）蛋白是外节形成所必需的，并且通过连接纤毛和IFT基因突变的蛋白质转运导致视网膜变性。IFT蛋白形成多亚基复合物，其通过驱动蛋白马达的作用沿连接纤毛中的微管沿着运输。我们将使用斑马鱼作为实验系统，因为斑马鱼的光感受器是很好的特点，建立技术存在于斑马鱼操纵基因表达和创造遗传嵌合体胚胎，我们有斑马鱼突变体的三个IFT基因。这些突变体表现出错误定位的视紫红质，破坏光感受器外节的形成，和光感受器变性。我们的目标是了解的功能和调控的IFT颗粒在纤毛蛋白质运输的感光细胞。具体而言，我们建议：1）通过光镜和电镜结合免疫组织化学方法观察3种IFT蛋白的结构和蛋白定位，探讨IFT蛋白突变对斑马鱼感光细胞外节发育的影响及其功能。2)研究Par-aPKC极性复合物对IFT颗粒组装和功能的调节。IFT颗粒和Par-aPKC复合物共定位在纤毛内，并且都与驱动蛋白-II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)通过使用荧光显微镜和延时共聚焦显微镜分析在IFT颗粒中组装的IFT-GFP融合蛋白的定位和运动，测量IFT复合物在IFT 57突变体中的稳定性和运动。4)通过使用吗啉代寡核苷酸在斑马鱼中产生Bardet-Biedl综合征表型，并通过光学和荧光显微镜评估感光细胞发育，确定感光细胞形态发生中对每种IFT蛋白的需求。由于感光细胞发育所需的蛋白质转运机制也在维持成年感光细胞中发挥作用，因此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.