Elucidating the Architecture and Mechanism of the Ciliary Gate

描述（由申请人提供）：纤毛是进化上古老的细胞器，可以移动并接收来自周围环境的信号，就像天线一样。根据细胞类型，纤毛可以传递机械感觉信号或发育信号，或推动细胞外液。不同功能的纤毛由不同的纤毛蛋白区分，纤毛基部的过渡区（transition zone，TZ）对纤毛蛋白的组成起着重要的调节作用。TZ功能的破坏会引起各种纤毛病变，包括多囊性肾、神经管缺陷和其他严重的发育障碍。尽管它在睫状体功能中起着核心作用，但TZ如何调节睫状体成分尚不清楚。为了解决纤毛生物学的主要空白，我将回答四个问题。1)睫状门的结构基础是什么？为了解决这个问题，我将使用三维随机光学重建显微镜（3D-STORM）和结构照明（SIM），以确定TZ架构原位培养的原代哺乳动物细胞。2)睫状门控的分子机制是什么？我将使用超分辨率显微镜和突变分析相结合，以评估如何访问纤毛蛋白TZ扰动后的不同。3)纤毛病变突变如何损害TZ结构？我将使用冷冻电子显微镜从嗜热四膜虫分离的TZs来解决纤毛病变如何改变TZs结构。4)具有不同功能的纤毛是否具有独特成分的TZs？我将分析TZ蛋白在四膜虫中的亚细胞定位，并对特化纤毛进行功能测定。我假设TZ内的不同结构包括不同的功能模块。我还试图阐明如何纤毛与不同的功能，如运动与感觉纤毛，多样化的特定TZ组件的差异表达，他们的角色可能。我的初步超分辨率显微镜研究揭示了几个TZ组件的精确位置，为TZ的结构提供了第一个见解。另外，我发现 TZ蛋白Tectonic 2和B 9d 1定位于四膜虫纤毛的不同亚群。进一步研究TZ蛋白可能调节四膜虫中交替纤毛的形成、信号传导或运动，将告知纤毛的特化是如何实现的。由于纤毛组织对其细胞功能至关重要，因此建立TZ结构将揭示 纤毛门控的结构基础，以及它在细胞信号传导和运动中的整体作用。重要的是，这项拟议的工作将阐明TZ结构缺陷导致纤毛病变的机制，TZ的精确分子图谱将作为设计睫状体疾病新治疗方法的框架。

DESCRIPTION (provided by applicant): Cilia are evolutionarily ancient organelles that can move and receive signals from their surroundings, much like antennae. Depending on the cell type, cilia can transduce mechanosensory signals or developmental signals, or propel extracellular fluid. Cilia with different functions are distinguished by distinct ciliary proteins.A region at the base of cilia called the transition zone (TZ) is critical for the regulation of ciliay protein composition. Disruption of TZ function causes a variety of ciliopathies, including polycystic kidneys, neural tube defects, and other severe developmental disorders. Despite its central role in ciliary function, how the TZ regulates ciliary composition is unknown. To address major gaps in cilia biology I will answer four questions. 1) What is the structural basis of the ciliary gate? To address this question, I will use three-dimensional Stochastic Optical Reconstruction Microscopy (3D-STORM) and Structured Illumination (SIM) to determine TZ architecture in situ in cultured primary mammalian cells. 2) What is the molecular mechanism of ciliary gating? I will use a combination of superresolution microscopy and mutational analysis to assess how access of ciliary proteins differs upon TZ perturbation. 3) How do ciliopathy mutations compromise TZ structure? I will use cryo-electron microscopy of TZs isolated from Tetrahymena thermophila to resolve how ciliopathies alter the TZ structure. 4) Do cilia with distinct functions have TZs with unique compositions? I will analyze the subcellular localization of TZ proteins in Tetrahymena and perform functional assays of specialized cilia. I hypothesize that distinct structures within the TZ comprise distinct functional modules. I also seek to elucidate how cilia with different functions, such as motile versus sensory cilia, diversify their roles potentially by differential expression of specific TZ components. My preliminary superresolution microscopy studies have revealed the precise location of several TZ components, providing the first insights into the architecture of the TZ. In addition, I have found that the TZ proteins Tectonic 2 and B9d1 localize to distinct subsets of cilia in Tetrahymena. Further investigation of TZ proteins that potentially regulate the formation, signaling, or motilit of alternate cilia in Tetrahymena will inform how specialization of cilia is achieved. Because cili organization is critical to its cellular function, establishing the TZ architecture will reveal the structural basis of ciliary gating, as well as its overall role in cell signaling and motility. Importantly, this proposed work will shed light on the mechanisms by which defects in TZ structure cause ciliopathies, and a precise molecular map of the TZ will serve as a framework for the design of novel treatments for ciliary diseases.