Structure and Function of Primary Cilia

初级纤毛的结构和功能

• 批准号: 8642654
• 负责人: Haixin Sui
• 金额: $ 25.95万
• 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/8642654
• 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; Human body; Image; Individual; Institutes; Kidney; Knowledge; Label; Lateral; Length; Life; Light; Link; Liver; Location; Maps; Medical; Membrane; Methodology; Methods; Microtubule-Associated Proteins; Microtubules; Molecular; Monitor; Morbidity - disease rate; Organelles; Organogenesis; Pancreas; Patients; Perinatal; 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; imaging modality; kidney cell; light microscopy; method development; particle; protein complex; public health relevance; 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器。它们的结构、组装和组成感觉蛋白的缺陷会导致各种各样的医学疾病和发育异常。许多关于初级纤毛的基本知识是不完整的，这是因为缺乏一种方便的方法来表征初级纤毛在其天然状态下的结构和组装过程。由于缺乏初级纤毛的三维(3D)结构，纤毛功能的分子机制研究相对滞后。对于由纤毛缺陷、药物刺激或环境变化引起的异常生长表型的表征，需要对野生型纤毛的组装和生长有详细的了解。如果没有这样的知识，很难确定最近发现的纤毛调节试剂是否能够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。该方案的目的是利用冷冻电子断层扫描(CRYO-ET)技术制作第一个详细的原生(玻璃体冷冻)初级纤毛的三维结构，并通过相关的光学和电子显微镜来确定初级纤毛的生长行为。我们突破性的方法开发为我们提供了有史以来第一个玻璃体冷冻的肾细胞初级纤毛的电子显微照片和断层照片。出乎意料的是，我们的研究还发现了睫状膜下的小囊泡。这一重要的发现将使我们能够检验初级纤毛的组装涉及膜小泡的鞭毛内转孢子(IFT)的假设。我们将通过以下两个具体目标开展研究：(1)利用冷冻-电子显微镜测定野生型肾脏初级纤毛的三维结构；(2)研究初级纤毛的生长行为，并确定生长是否与纤毛内小泡的IFT有关。这项拟议的研究具有非常重要的意义，因为它不仅将提供初级纤毛的第一个详细的自然结构，而且还将建立肾脏细胞的生长行为。我们将测试一个关于纤毛内小泡功能作用的重要假说，这可能会使我们对膜成分如何靶向纤毛的理解发生范式转变。我们的研究将是系统研究初生纤毛的第一步，以便了解各种纤毛蛋白或其复合体的作用。此外，在拟议的研究过程中开发的方法将为未来对初级纤毛的研究提供一个新的平台。例如，该平台可以用于研究睫毛病理动物模型的原代细胞或囊性疾病患者的活检标本，它将有助于VITR药物的筛选和治疗的开发。