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、贾第鞭毛虫和纳格里原虫。纤毛组装需要通过纤束内运输复合物和分子马达将前体从细胞质池运输到组装位点，但该系统的货物识别尚不清楚。此外，大多数纤毛蛋白作为更大复合物的一部分组装，指导这些复合物预组装的机制尚不清楚。这些步骤对于仅作为感觉触角的纤毛轴突的形成以及纤毛中以动力蛋白为基础的运动至关重要。在这里，我们利用模式生物莱茵衣藻（Chlamydomonas reinhardtii）中影响运动纤毛中丰富复合物（多亚单位轴突外臂动力马达）的预组装和运输的突变，揭示了每个步骤的基本机制，并表征了这一途径中重要的基因。在Aim 1中，我们将利用阻断这一步骤的三种突变（pf13， pf22和oda7）来表征动力蛋白复合物在细胞质中的预组装。我们将通过生物化学方法分离含有每个装配位点基因产物的复合物，从而验证这些蛋白质作为轴突动力蛋白折叠和/或预组装的共同伴侣的假设。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。为了理解动力蛋白的运输，我们将在不同条件下对货物和适配器进行免疫定位，包括鞭毛组装，鞭毛拆卸，以及在细胞中携带改变组装过程的各种突变和突变组合。在Aim 3中，我们将利用一种新的细胞质复合物组装能力的体外检测方法，验证ODA5、ODA8和ODA10位点的产物形成一个复合物的假设，该复合物在后期作用，使运输的外臂动力蛋白能够与轴突结合。我们的研究结果将确定新的人类疾病候选基因，并描述轴突蛋白组装的保守步骤。

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.