Characterizing three-dimensional structures at the base of primary and motile cilia using cryo-electron tomography

使用冷冻电子断层扫描表征初级纤毛和活动纤毛底部的三维结构

• 批准号: 10583292
• 负责人: Justine Marie Pinskey
• 金额: $ 0.25万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10583292
• 关键词: 3-Dimensional; Address; Affect; Architecture; Area; Binding; Biological; Biological Process; Cell surface; Cells; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Complex; Cryo-electron tomography; Data; Defect; Development; Diagnosis; Disease; Electron Microscopy; Embryo; Eukaryotic Cell; Fibroblasts; Flagella; Foundations; Freezing; Future; Gallium; Goals; Hair; Health; Homeostasis; Human; Image; Individual; Institution; Ions; Kidney; Knowledge; Location; Maps; Microscopy; Molecular; Molecular Structure; Mus; Mutate; Mutation; Nature; Organelles; Pattern; Play; Preparation; Process; Proteins; Protocols documentation; Research; Research Personnel; Resolution; Sampling; Signal Transduction; Structural defect; Structure; Therapeutic Intervention; Thick; Thinness; Training; Work; base; cell motility; ciliopathy; cilium motility; diagnostic tool; experimental study; human disease; improved; innovation; insight; intercellular communication; interest; kinetosome; light microscopy; mutant; prevent; three dimensional structure; trafficking

ABSTRACT Cilia are nearly ubiquitous among eukaryotic cells and regulate important processes including motility, sensing, and signaling. In humans, both motile and immotile (primary) cilia are critical for development and homeostasis, with disruptions in ciliary function leading to a group of diseases known as ciliopathies. While the three- dimensional structure of the axoneme of motile cilia has been well-characterized, the molecular structures of primary cilia, as well as those that comprise the base of cilia (the basal body and transition zone) remain poorly understood. Taking aim at these gaps, I will use innovative approaches, including cryo-correlative light and electron microscopy, cryo-focused ion beam milling, cryo-electron tomography, and subtomogram averaging to generate high-resolution structures of primary cilia from mouse embryonic fibroblasts and the basal body/transition zone from Chlamydomonas cells. Furthermore, I will characterize the location, structure and molecular interactions of the transition zone protein Nephrocystin-4 (NPHP4), which helps regulate molecular trafficking into and out of the cilium and causes human ciliopathies when mutated. Specifically, I will visualize and compare ciliary structures from wild type and nphp4-null Chlamydomonas and precisely analyze the size and location of nphp4-mutant structural defects. This research will further our understanding of the three- dimensional architecture and function of cilia and reveal structural changes underlying ciliary disease mechanisms.

抽象的 纤毛在真核细胞中几乎无处不在，并调节重要过程，包括运动，感应， 和信号。在人类中，机动和immotile（主要）纤毛对于发展和体内平衡至关重要， 睫状功能的破坏导致一组称为纤毛病的疾病。而三人 纤毛的轴突的尺寸结构已得到充分表征，即分子结构 原发性纤毛，以及组成纤毛基础（基体和过渡区）的纤毛纤维 理解。为了瞄准这些差距，我将使用创新的方法，包括冷冻相关的光和 电子显微镜，以冷冻关注的离子束铣削，冷冻电子层析成像和亚图平均 从小鼠胚胎成纤维细胞和基底产生原代纤毛的高分辨率结构 衣原体细胞的身体/过渡区。此外，我将表征位置，结构和 过渡区蛋白质肾环胞固醇4（NPHP4）的分子相互作用，有助于调节分子 贩运纤毛进出，并在突变时引起人类纤毛病。具体来说，我将可视化 并比较野生型和nphp4-null衣原体的睫状结构，并精确分析大小 NPHP4突变结构缺陷的位置。这项研究将进一步我们对三个的理解 纤毛的维度结构和功能，并揭示睫毛疾病的结构变化 机制。