The regulation of ciliary motility by an axonemal PP2A phosphatase

轴丝 PP2A 磷酸酶对纤毛运动的调节

• 批准号: 8180484
• 负责人: Candice A Elam
• 金额: $ 1.95万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8180484
• 关键词: Address; Affinity; Antibodies; Binding; Biological Models; Blindness; Catalytic Domain; Cells; Cerebral Ventricles; Chemicals; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Co-Immunoprecipitations; Column Chromatography; Complex; Data; Development; Disease; Docking; Dominant-Negative Mutation; Dynein ATPase; Electron Microscopy; Epithelium; Failure; Family; Fertility; Flagella; Fluorescence Microscopy; Gene Expression; Gene Mutation; Genetic Models; Goals; Human; Hydrocephalus; Immunoprecipitation; Individual; Infertility; Kidney; Lead; Learning; Left; Libraries; Liquid substance; Mammalian Oviducts; Microscopy; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular Motors; Movement; Mutation; Obesity; Organ; Organism; Paralysed; Pattern Formation; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Photoreceptors; Plasmids; Play; Polycystic Kidney Diseases; Positioning Attribute; Post-Translational Protein Processing; Precipitation; Primary Ciliary Dyskinesias; Protein Binding; Protein phosphatase; Proteins; Proteomics; Publishing; RNA Interference; Regulation; Respiratory System; Role; Sensory; Shapes; Signal Transduction; Slide; Sodium Chloride; Specificity; Spectrophotometry; Structure; Substrate Specificity; Sucrose; Swimming; System; Techniques; Testing; Time; Tissues; Transfection; Tubulin; WD Repeat; Work; arm; base; cell motility; crosslink; design; light microscopy; male; member; molecular phenotype; mutant; protein complex; protein protein interaction; research study; restoration; scaffold; sperm cell; stem; tool

DESCRIPTION (provided by applicant): The overall goal of my thesis is to define the mechanisms that regulate ciliary/flagellar motility. In particular, my focus is on the regulation of dynein-driven microtubule sliding by phosphorylation. The work is important because cilia are responsible for motile functions (e.g. movement of sperm, fluid movement along epithelia, such as in the respiratory system, oviducts, or brain ventricles) and sensory functions (e.g. the connecting cilia of photoreceptors, the mechano-sensitive primary cilia of the kidney, etc). Failure in assembly or function can lead to a wide range of diseases in humans including, polycystic kidney, blindness, infertility in males, and primary ciliary dyskinesia. Thus it is important to understand how cilia are assembled and how dynein-driven motility is regulated. My project is focused on the protein phosphatase PP2A, which based on previous studies and my new preliminary data, is located in the ciliary axoneme and controls dynein activity. PP2A is made up of three subunits, the anchoring A-subunit, the catalytic C-subunit, and the regulatory B-subunit. The B-subunit is responsible for providing the phosphatase with substrate specificity and sub-cellular localization. This proposal is designed to test the hypothesis that PP2A is localized and anchored in the axoneme by a regulatory B-subunit to modulate phosphatase activity. The following aims are designed to test this hypothesis: In specific aim 1,1 propose to use immuno-fluorescence and electron microscopy to test the hypothesis that the B-subunit of PP2A is localized to the outer doublet microtubules in position to regulate inner arm 11 dynein, the dynein that appears to be critical for control of microtubule sliding and ciliary bending. For aim 2, using immunoprecipitation, chemical crosslinking, and mass spectrophotometry, I will test the hypothesis that the B-subunit, in addition to binding the A and C-subunits, interacts with other axonemal proteins that serve as a docking complex to anchor and localize it within the axoneme near 11 dynein. Finally, in aim 3,1 will address the function of the B-subunit by assessing motility and molecular phenotypes of mutants defective in B-subunit gene expression. Together, these data will comprehensively test the functional role of PP2A in the ciliary axoneme. Moreover, it is also possible that we will define a general role for PP2A B-subunits in targeting the phosphatase to microtubule dependent functions.

描述（由申请人提供）：我论文的总体目标是定义调节纤毛/鞭毛运动的机制。特别是，我的重点是通过磷酸化对动力蛋白驱动的微管的调节。这项工作很重要，因为纤毛负责运动功能（例如精子的运动，沿上皮的流动运动，例如呼吸系统，产卵或脑室的运动）和感觉功能（例如，光感应者的纤毛连接纤毛，纤毛的纤毛，小子机械敏感的原​​发性纤毛等）。组装或功能的失败会导致人类的各种疾病，包括多囊性肾脏，失明，男性不育症和原发性睫状运动障碍。因此，重要的是要了解纤毛的组装方式以及如何调节动力蛋白驱动的运动。我的项目集中在蛋白质磷酸酶PP2A上，该蛋白质磷酸酶PP2A基于先前的研究和我的新初步数据，位于纤毛轴突中，并控制动力蛋白活性。 PP2A由三个亚基组成，即锚定A-亚基，催化C-亚基和调节B-Subunit。 B-Subunit负责为磷酸酶提供底物特异性和亚细胞定位。该提案旨在检验以下假设：PP2A通过调节B-Subunit定位并锚定在轴突中以调节磷酸酶活性。以下目的旨在检验这一假设：在特定目的中，1,1提议使用免疫荧光和电子显微镜检验以下假设：PP2A的B-子元素位于外部双倍微管上，以适当地调节内部臂11型蛋白，对微动物的控制至关重要。对于AIM 2，使用免疫沉淀，化学交联和质量分光光度法，我将测试以下假设：除了结合A和C-亚基外，B-亚基与其他轴突蛋白相互作用，这些轴突蛋白与其他轴基蛋白相互作用，这些轴突蛋白可以用作固定在11 dynein接近11 dynein中的对接配合物并将其定位在轴突中。最后，在AIM 3,1中，将通过评估B-Subunit基因表达中有缺陷的突变体的运动性和分子表型来解决B-亚基的功能。总之，这些数据将全面测试PP2A在睫状轴突中的功能作用。此外，我们也可能会定义PP2A B-亚基在靶向磷酸酶对微管依赖性函数方面的一般作用。