Molecular and Genetic Analysis of Flagellar Dyneins

描述(申请人提供)：本提案重点介绍了真核纤毛和鞭毛的组装和维护。这些细胞器几乎存在于每个人类细胞中，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折磨智人的生物，如锥虫、利什曼原虫、疟原虫、贾第鞭毛虫和奈格勒原虫。纤毛组装需要通过鞭毛内转运复合体和分子马达将前体从细胞质池运输到组装位置，但该系统对货物的识别仍不清楚。此外，大多数纤毛蛋白作为较大复合体的一部分组装，而指导这些复合体预组装的机制尚未确定。这些步骤对于纤毛中仅起感觉触角作用的轴丝的形成以及对其基于动力蛋白的运动至关重要的纤毛是必不可少的。在这里，我们使用模式生物莱茵衣藻的突变来揭示每一步的基本机制，并表征在这一途径中重要的基因。在目标1中，我们将利用阻止这一步骤的三个突变(pf13、pf22和oda7)来表征动力蛋白复合体在细胞质中的预组装。我们将通过生化方法分离包含每个组装位点基因产物的复合体，以检验这些蛋白质作为轴丝动力蛋白折叠和/或预组装的辅助伴侣的假设。目标2中提出的实验将表征IFT运输机械对货物的识别，并测试ODA16作为动力蛋白运输货物适配器的作用。为了了解动力蛋白的运输，我们将在不同的条件下对货物和接头进行免疫定位，包括在鞭毛组装、鞭毛分解以及含有各种突变和突变组合的细胞中，这些突变和突变组合会改变组装过程。在目标3中，我们将利用一种新的细胞质复合体组装能力的体外测试方法，测试ODA5、ODA8和ODA10基因座的产物形成一个复合体的假设，该复合体在后期作用，使运输的外臂动力蛋白能够与轴丝结合。我们的结果将确定新的人类疾病候选基因，并表征轴丝蛋白组装中的保守步骤。 公共卫生相关性：这项提案侧重于组装和维持真核生物纤毛和鞭毛所需的基因。这些细胞器几乎在每个人类细胞上都能找到，它们的缺失会导致不育和危及生命的疾病，包括呼吸道和肾脏疾病。它们在许多引起人类疾病的原生生物的生命周期中也是必不可少的，例如锥虫病、利什曼病、疟疾、贾第鞭毛虫病和旋毛虫病。

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.