Structure and Function of Primary Cilia

初级纤毛的结构和功能

• 批准号: 8438723
• 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/8438723
• 关键词: Animal Model; Behavior; Biopsy Specimen; Cells; Cilia; Complex; Cystic Kidney Diseases; Defect; Degenerative Disorder; 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（由申请人提供）：初级纤毛是参与许多生理过程的感觉细胞器。它们的结构、组装和感觉蛋白组分的缺陷导致各种各样的医学疾病和发育异常。由于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，关于初级纤毛的许多基础知识是不完整的。由于缺乏初级纤毛的参考三维（3D）结构，纤毛功能的分子机制的研究是滞后的。野生型纤毛及其生长的组装的详细知识是必要的，以表征异常生长表型，是由于纤毛缺陷，药物刺激或环境变化。如果没有这些知识，就很难确定最近鉴定的纤毛发生调节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。 该提案的目的是使用冷冻电子断层扫描（cryo-ET）产生第一个详细的原生（玻璃体冷冻）初级纤毛的3D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。我们突破性的方法发展为我们提供了有史以来第一个电子显微镜和断层摄影的玻璃冷冻初级纤毛的肾细胞。出乎意料的是，我们的研究还发现了睫状膜下的小泡。这一重要发现将使我们能够测试的假设，初级纤毛的组装涉及膜泡的鞭毛内转运（IFT）。我们将通过以下两个具体目标进行我们的研究：（1）我们将通过cryo-ET确定野生型肾脏初级纤毛的3D结构;（2）我们将研究初级纤毛的生长行为，并确定生长是否与睫状囊泡的IFT有关。这项研究非常重要，因为它不仅将提供第一个详细的原生纤毛结构，而且还将建立肾细胞中的细胞生长行为。我们将测试一个重要的假设睫状囊泡的功能作用，这可能会引入一个范式转变，在我们的理解膜组件如何靶向纤毛。我们的研究将是连续研究的第一步，以系统地表征初级纤毛，以了解各种纤毛蛋白或其复合物的作用。此外，在拟议的研究过程中开发的方法将为初级纤毛的未来研究提供一个新的平台。该平台可用于例如研究来自纤毛病理学动物模型或来自囊性疾病患者的活检标本的原代细胞，并且它将有助于体外药物筛选和治疗开发。