Elucidating the Architecture and Mechanism of the Ciliary Gate

阐明睫状门的结构和机制

• 批准号: 8835431
• 负责人: GALO GARCIA
• 金额: $ 5.42万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835431
• 关键词: Address; Affect; Affinity; Architecture; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Biology; Brain; Carrier Proteins; Cell physiology; Cells; Cilia; Color; Complex; Cryoelectron Microscopy; Cystic kidney; Defect; Development; Diabetes Mellitus; Disease; Epithelial Cells; Extracellular Fluid; Genes; Genetic; Image; In Situ; Individual; Investigation; Knock-out; Life; Light; Lighting; Location; Mammalian Cell; Maps; Mass Spectrum Analysis; Membrane Proteins; Methods; Microscopy; Molecular; Mus; Mutant Strains Mice; Mutation; Nephronophthisis; Neural Tube Defects; Obesity; Optics; Oral; Organelles; Organism; Play; Polycystic Kidney Diseases; Polydactyly; Property; Proteins; Regulation; Relative (related person); Retinal Degeneration; Role; Sensory; Signal Transduction; Specificity; Spinal Cord; Structure; Surface; Tertiary Protein Structure; Testing; Tetrahymena; Tetrahymena thermophila; Tissues; Wild Type Mouse; Work; base; cell motility; cell type; ciliopathy; cilium biogenesis; design; developmental disease; differential expression; feeding; insight; intercellular communication; mutant; novel; paralogous gene; polypeptide; public health relevance; reconstruction; tool

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.

描述（由申请人提供）：纤毛是进化上古老的细胞器，可以移动和接收来自周围环境的信号，很像触角。根据细胞类型的不同，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来区分。纤毛基部的过渡区（TZ）对纤毛蛋白质组成的调节至关重要。TZ功能的破坏可引起多种纤毛病，包括多囊肾、神经管缺陷和其他严重的发育障碍。尽管TZ在纤毛功能中起着核心作用，但其如何调节纤毛成分尚不清楚。为了解决纤毛生物学的主要空白，我将回答四个问题。1)纤毛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）来原位确定培养的原代哺乳动物细胞的TZ结构。2)纤毛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的组合来评估纤毛蛋白在TZ扰动下的访问如何不同。3)纤毛病突变如何损害TZ结构？我将使用从嗜热四膜虫中分离的TZ的低温电子显微镜来解决纤毛病如何改变TZ结构。4)功能不同的纤毛是否具有独特成分的TZs ？我将分析四膜虫中TZ蛋白的亚细胞定位，并对特殊纤毛进行功能分析。我假设TZ内不同的结构包含不同的功能模块。我还试图阐明具有不同功能的纤毛，如运动纤毛和感觉纤毛，如何通过特定TZ成分的差异表达来潜在地多样化其作用。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