Structure and Function of Primary Cilia

初级纤毛的结构和功能

• 批准号: 9012832
• 负责人: Haixin Sui
• 金额: $ 26.12万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9012832
• 关键词: Animal Model; Behavior; Biopsy Specimen; Cells; Cilia; Complex; Cystic Kidney Diseases; Defect; Development; Disease; Electron Microscopy; Electrons; Environment; Epithelial Cells; Freezing; Functional disorder; Genetic; Goals; Growth; Health; Human body; Image; Individual; Institutes; Kidney; Knowledge; Label; Lateral; Length; Light; Link; Liver; Location; Maps; Medical; Membrane; Methodology; Methods; Microtubule-Associated Proteins; Microtubules; Molecular; Monitor; Organelles; Organogenesis; Pancreas; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiological Processes; Play; Population Distributions; Preclinical Drug Evaluation; Process; Proteins; Public Health; Reagent; Research; Resolution; Retinal Degeneration; Role; Sensory; Side; Staging; Stimulus; Structure; Syndrome; Testing; Three-Dimensional Image; Time; Tomogram; Transmembrane Transport; Vesicle; base; cell type; ciliopathy; cilium biogenesis; developmental disease; drug development; electron tomography; environmental change; kidney cell; light microscopy; live cell imaging; method development; particle; perinatal morbidity; protein complex; therapy development; three dimensional structure

DESCRIPTION (provided by applicant): Primary cilia are sensory organelles involved in many physiological processes. Defects in their structure, assembly, and component sensory proteins cause a wide variety of medical disorders and developmental abnormities. Much of the fundamental knowledge about primary cilia is incomplete, due to lack of a convenient methodology to characterize the structure and assembly processes of primary cilia in their native state. In the absence of a reference three-dimensional (3D) structure of primary cilia, studies on molecular mechanisms of ciliary functions are lagging. A detailed knowledge of the assembly of wild-type cilia and their growth are necessary to characterize abnormal-growth phenotypes that are due to ciliary defects, drug stimuli or environmental changes. Without such knowledge it is difficult to determine if the recently identified regulatory reagents of ciliogeness can restore or maintain normal cilium structure and if they can be used for potential drug development. The objectives of this proposal are to produce the first detailed 3D structure of native (vitreously frozen) primary cilia using cryo-electron tomography (cryo-ET), and to determine the growth behavior of primary cilia by correlative light and electron microscopy. Our ground-breaking method development has provided us the first- ever electron micrographs and tomograms of vitreously frozen primary cilia of kidney cells. Unexpectedly, our study also revealed small vesicles immediately beneath the ciliary membrane. This important finding will allow us to test the hypothesis that the assembly of primary cilia involves intraflagellar transpor (IFT) of membrane vesicles. We will carry out our research through the following two specific aims: (1) We will determine the 3D structure of the wild-type kidney primary cilium by cryo-ET; (2) We will study the growth behavior of primary cilia, and will determine whether growth is related to IFT of intra-ciliary vesicles. The proposed research is highly significant because it wil not only provide the first detailed native structure of primary cilia, but it will also establish teir growth behavior in kidney cells. We will test an important hypothesis about the functional roles of intra-ciliary vesicles that would potentially introduce a paradigm shift in our understanding about how membrane components target to the cilium. Our study will be the first step in a continuum of research to systematically characterize primary cilia in order to understand the roles of various ciliary proteins or their complexes. Furthermore, the methods developed during the course of the proposed study will provide a new platform for future research on primary cilia. This platform can be used, for example, to study primary cells from ciliopathological animal models or from biopsy specimens of patients with cystic diseases, and it will contribute to in vitr drug screening and therapy development.

描述（申请人提供）：初级纤毛是参与许多生理过程的感觉细胞器。它们的结构、组装和组成感觉蛋白的缺陷导致各种各样的医学障碍和发育异常。由于缺乏一种方便的方法来表征原生状态下初级纤毛的结构和组装过程，许多关于初级纤毛的基础知识是不完整的。由于缺乏初级纤毛的三维结构参考，对纤毛功能的分子机制研究滞后。对野生型纤毛的组装及其生长的详细了解对于表征由纤毛缺陷、药物刺激或环境变化引起的异常生长表型是必要的。如果没有这些知识，很难确定最近发现的纤毛调节试剂是否可以恢复或维持正常的纤毛结构，以及它们是否可以用于潜在的药物开发。本提案的目的是利用冷冻电子断层扫描（cryo-ET）产生原生（玻璃体冷冻）初级纤毛的第一个详细的3D结构，并通过相关的光学和电子显微镜确定初级纤毛的生长行为。我们突破性的方法发展为我们提供了有史以来第一个玻璃体冷冻肾细胞原代纤毛的电子显微照片和断层摄影。出乎意料的是，我们的研究还发现了纤毛膜下的小泡。这一重要发现将使我们能够验证初级纤毛的组装涉及膜囊的鞭毛内转运（IFT）的假设。我们将通过以下两个具体目的开展我们的研究：(1)我们将通过冷冻et确定野生型肾脏初级纤毛的三维结构；(2)我们将研究初级纤毛的生长行为，确定生长是否与纤毛内小囊的IFT有关。这项研究具有重要的意义，因为它不仅提供了初级纤毛的第一个详细的天然结构，而且还将建立它们在肾细胞中的生长行为。我们将测试一个关于纤毛内囊泡功能作用的重要假设，这可能会在我们对膜成分如何靶向纤毛的理解中引入范式转变。我们的研究将是系统表征初级纤毛的连续研究的第一步，以了解各种纤毛蛋白或其复合物的作用。此外，在研究过程中开发的方法将为未来的初级纤毛研究提供一个新的平台。例如，该平台可用于研究来自纤毛病理动物模型或囊性疾病患者活检标本的原代细胞，并将有助于体外药物筛选和治疗开发。