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: 10259676
• 负责人: Justine Marie Pinskey
• 金额: $ 6.64万
• 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/10259676
• 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; Diagnostic; 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; experimental study; human disease; improved; innovation; insight; intercellular communication; interest; kinetosome; light microscopy; mutant; prevent; three dimensional structure; tool; 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.

摘要 纤毛在真核细胞中几乎普遍存在，并调节重要的过程，包括运动，感觉， 和信号。在人类中，活动和不活动的（初级）纤毛对于发育和体内平衡都是至关重要的， 纤毛功能的破坏导致一组称为纤毛病的疾病。当这三个- 运动纤毛轴丝的三维结构已经得到很好的表征， 初级纤毛以及构成纤毛基部的纤毛（基体和过渡区）仍然很差 明白针对这些差距，我将使用创新的方法，包括低温相关光， 电子显微镜、低温聚焦离子束铣削、低温电子断层扫描和亚断层图像平均， 从小鼠胚胎成纤维细胞产生初级纤毛的高分辨率结构， 衣原体细胞的体/过渡区。此外，我将描述的位置，结构和 过渡区蛋白肾囊蛋白4（NPHP 4）的分子相互作用，有助于调节分子 运输进出纤毛，突变时引起人类纤毛病。具体来说，我会想象 比较野生型和nphp 4缺失衣原体的纤毛结构， 和nphp 4突变体结构缺陷的定位。这项研究将进一步加深我们对这三个- 纤毛的三维结构和功能，并揭示纤毛疾病背后的结构变化 机制等