Molecular and Genetic Analysis of Flagellar Dyneins

描述（由申请人提供）：本提案侧重于真核纤毛和鞭毛的组装和维持。这些细胞器在几乎每个人类细胞上都有发现，并且在大多数其他真核生物的生命周期中也是必不可少的，包括那些作为致病生物体折磨智人的生物，如锥虫属，利什曼原虫属，疟原虫属，贾第虫属和耐格里属。纤毛装配需要运输的前体从细胞质池组装网站的鞭毛内运输复合物和分子马达，但货物识别这一系统仍然未知。此外，大多数纤毛蛋白组装为较大复合物的一部分，并且指导这些复合物的预组装的机制尚未表征。这些步骤是必不可少的纤毛中的轴丝的形成，只作为感觉天线，以及在纤毛重要的动力蛋白为基础的运动。在这里，我们使用的模式生物莱茵衣藻的突变，影响预组装和运输丰富的复杂的运动纤毛，多亚基轴丝外臂动力蛋白电机，揭示每个步骤的基本机制和特征基因在这一途径中的重要性。在目标1中，我们将利用阻止这一步骤的三个突变（pf 13，pf 22和oda 7）来表征细胞质中动力蛋白复合物的预组装。我们将测试的假设，这些蛋白质作为共分子伴侣的折叠和/或轴丝动力蛋白的预组装分离复合物含有每个组装位点的基因产物，通过生化方法。目标2中提出的实验将表征IFT运输机械的货物识别，并测试ODA 16作为动力蛋白运输的货物适配器的作用。为了了解动力蛋白运输，我们将免疫定位货物和适配器在不同的条件下，包括在鞭毛组装，鞭毛拆卸，并在细胞窝藏各种突变和突变体组合，改变装配过程。在目标3中，我们将测试的假设，ODA 5，ODA 8和ODA 10位点的产品形成一个复杂的行为，在后期步骤，使运输外臂动力蛋白主管轴丝结合，利用一种新的体外测定组装能力的细胞质复合物。我们的研究结果将确定新的人类疾病候选基因和轴丝蛋白组装的保守步骤的特点。 公共卫生相关性：这项提案的重点是真核生物纤毛和鞭毛的组装和维持所需的基因。这些细胞器几乎存在于每一个人类细胞中，它们的缺失会导致不育和危及生命的疾病，包括呼吸系统和肾脏疾病。它们在许多引起人类疾病的原生动物生物体的生命周期中也是必不可少的，如锥虫病、利什曼病、疟疾、贾第虫病和疟疾。

DESCRIPTION (provided by applicant): This proposal focuses on the assembly and maintenance of eukaryotic cilia and flagella. These organelles are found on nearly every human cell and are also essential in the life cycles of most other eukaryotic organisms, including those that afflict Homo sapiens as disease causing organisms, such as Trypanosoma, Leishmania, Plasmodium, Giardia and Naegleria. Ciliary assembly requires transport of precursors from cytoplasmic pools to assembly sites by intraflagellar transport complexes and molecular motors, but cargo recognition by this system remains unknown. In addition, most ciliary proteins assemble as parts of larger complexes, and mechanisms that direct pre-assembly of these complexes are uncharacterized. These steps are essential for the formation of axonemes in cilia that act only as sensory antennae as well as in cilia important for their dynein-based motility. Here we use mutations in the model organism Chlamydomonas reinhardtii that affect pre-assembly and transport of an abundant complex in motile cilia, the multi-subunit axonemal outer arm dynein motor, to uncover basic mechanisms of each step and characterize genes important in this pathway. In Aim 1 we will characterize the pre-assembly of dynein complexes in the cytoplasm, taking advantage of three mutations (pf13, pf22 and oda7) that block this step. We will test the hypothesis that these proteins act as co-chaperones for folding and/or pre-assembly of axonemal dyneins by isolating complexes containing each assembly locus gene product through biochemical approaches. Experiments proposed in Aim 2 will characterize cargo recognition by the IFT transport machinery and test the role of ODA16 as a cargo adaptor for dynein transport. To understand dynein transport we will immunolocalize cargo and adaptor under varying conditions, including during flagellar assembly, flagellar disassembly, and in cells harboring various mutations and mutant combinations that alter the assembly process. In Aim 3 we will test the hypothesis that products of the ODA5, ODA8 and ODA10 loci form a complex that acts at a late step to make transported outer arm dynein competent for axonemal binding, taking advantage of a new in vitro assay for assembly-competency of cytoplasmic complexes. Our results will identify new human disease gene candidates and characterize conserved steps in assembly of axonemal proteins. PUBLIC HEALTH RELEVANCE: This proposal focuses on genes needed for the assembly and maintenance of eukaryotic cilia and flagella. These organelles are found on nearly every human cell and their absence results in sterility and life-threatening conditions including respiratory and kidney disease. They are also essential in the life cycles of many protozoan organisms that cause human diseases, such as trypanosomiasis, leishmaniasis, malaria, giardiasis, and trichosomiasis.