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

摘要 纤毛在真核细胞中几乎无处不在，调节着重要的过程，包括运动，感觉， 和信号。在人类中，活动的和静止的(初级)纤毛对发育和动态平衡都是至关重要的， 睫毛功能的紊乱会导致一组被称为纤毛疾病的疾病。而三个人- 运动纤毛的轴丝的空间结构已被很好地表征，其分子结构 初级纤毛以及构成纤毛底部(基底体和过渡区)的纤毛仍然很差。 明白了。针对这些差距，我将使用创新的方法，包括低温相关光和 电子显微镜、低温聚焦离子束研磨、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和基质细胞中获得高分辨率的原生纤毛结构 衣藻细胞的体部/过渡区。此外，我将描述位置、结构和 帮助调节分子的过渡区蛋白Nephrocystin-4(NPHP4)的分子作用 当突变时，贩运进和出纤毛并导致人类纤毛病。具体地说，我将把 比较野生型和NPHP4阴性衣藻的纤毛结构，并精确分析其大小 以及NPHP4突变结构缺陷的位置。这项研究将加深我们对这三个问题的理解-- 纤毛的空间结构和功能，揭示睫状结构疾病潜在的结构变化 机械装置。

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.