A Role for the Calcium activated Chloride Channel TMEM16a in Primary Ciliogenesis

钙激活氯离子通道 TMEM16a 在初级纤毛发生中的作用

• 批准号: 9761117
• 负责人: Skylar Fisher
• 金额: $ 4.5万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761117
• 关键词: Affect; Apical; Bardet-Biedl Syndrome; Biogenesis; Biological Process; Biology; Biophysics; Blindness; CDC42 gene; CRISPR/Cas technology; Calcium; Calcium Signaling; Cell Differentiation process; Cell Survival; Cell membrane; Cell physiology; Cells; Cellular Structures; Centrioles; Chloride Channels; Cilia; Clinical; Collection; Defect; Development; Disease; Ductal Epithelial Cell; Electrophysiology (science); Embryo; Environment; Esthesia; Event; Family; Fluorescence Resonance Energy Transfer; Functional disorder; Genes; Goals; Image; Immunofluorescence Immunologic; In Vitro; Interphase Cell; Ion Channel; Joubert syndrome; Knock-out; Knockout Mice; Knowledge; Label; Length; Limb Bud; Link; Lipids; MEL Gene; Maintenance; Measures; Microtubules; Mitosis; Mitotic spindle; Modification; Molecular; Mothers; Motivation; Mus; Mutation; Neonatal; Neural tube; Obesity; Organelles; Output; Pathway interactions; Pharmacology; Physiological; Physiological Processes; Play; Process; Proteins; Quantitative Reverse Transcriptase PCR; Regulation; Research; Role; SHH gene; Sensory; Side; Signal Pathway; Signal Transduction; Structure; Symptoms; Techniques; Testing; Tissues; Transfection; Tubulin; Vertebral column; Vesicle; WNT Signaling Pathway; Western Blotting; Work; appendage; base; body system; cellular imaging; ciliopathy; cilium biogenesis; clinical phenotype; experimental study; human disease; improved; in vivo; insight; kidney dysfunction; knock-down; live cell microscopy; malformation; migration; mutant; novel; sensor; smoothened signaling pathway; trafficking

PROJECT SUMMARY The primary cilium, an apical cellular appendage akin to a cellular antenna, is involved in a variety of physiological processes including development, proliferation, and cell survival. Mutations in many genes linked to ciliary proteins cause a family of human diseases termed ciliopathies which include Joubert Syndrome and Bardet- Biedl syndrome. Evidencing the ubiquitous function of the primary cilium, these diseases present diverse clinical phenotypes affecting many of the body’s organ systems. As fundamental signaling hubs with distinct protein and lipid compositions relative to the rest of the cell, primary cilia are highly regulated in both their formation and function. Ion channels represent a unique class of proteins which regulate signaling events in many cellular processes, but are only beginning to be understood and appreciated in the context of the cilium. We are using a combination of electrophysiology and cell imaging to study the role of the calcium activated chloride channel ANO1 in regulating primary ciliogenesis and ciliary signaling. We recently found that Ano1 knock out results in fewer and shorter cilia compared to cells expressing ANO1. Furthermore, knock out of Ano1 in mice results in neonatal lethality due to tissue malformation mirroring classical ciliopathy hallmarks. We also recently characterized a novel cellular structure, the nimbus, which contains ANO1 and appears spatio-temporally related to ciliogenesis. We will test the hypothesis that ANO1 plays a role in regulating cilium formation and function. First, we will characterize the localization and activity of ANO1 during ciliogenesis. These experiments when during ciliogenesis ANO1 activity is important, and what step(s) of ciliogenesis ANO1 regulates. Finally, we will quantify ciliary signaling outputs in the presence and absence of ANO1. This work will allow us to elucidate the role of ANO1 in primary ciliogenesis, the functional consequence of loss of ANO1, and provide insight into primary cilium ion channel biology overall. Understanding the molecular pathways will provide valuable insights into the study of calcium activated chloride channels, and ion channels in the primary cilium. This fundamental knowledge can then be applied to primary cilia in the context of organismal development and ciliopathies and may provide new insight into pathways and targets for the treatment of these diseases.

项目摘要 原发性纤毛是类似于细胞天线的顶端细胞附加物，参与了多种生理 包括发育，增殖和细胞存活在内的过程。与睫状相关的许多基因的突变 蛋白质引起的人类疾病家族称为纤毛病，其中包括乔伯特综合征和Bardet- Biedl综合征。这些疾病呈现了原发性纤毛的无处不在功能，这些疾病呈现潜水员的临床 影响人体许多器官系统的表型。作为具有不同蛋白质和 脂质组成相对于细胞的其余部分，主要的纤毛在其形成和 功能。离子通道代表独特的蛋白质类别，该蛋白质调节许多细胞中的信号事件 过程，但才在纤毛的背景下才开始被理解和欣赏。我们正在使用 电生理学和细胞成像的组合研究钙活化的氯化物通道的作用 调节原发性纤毛发生和纤毛信号传导时的ANO1。最近，我们发现Ano1淘汰了结果 与表达ANO1的细胞相比，纤毛较少和较短。此外，从小鼠中敲出ANO1会导致 由于组织畸形引起的新生儿致死性，反映了经典的纤毛疗法标志。我们最近也 表征了一种新型的细胞结构，该结构包含ANO1，并且看起来是空间相关的 纤毛发生。我们将测试ANO1在调节纤毛形成和功能中起作用的假设。 首先，我们将表征纤毛发生过程中ANO1的定位和活性。这些实验何时 在纤毛发生过程中，ANO1活性很重要，以及纤毛生成的步骤ano1调节。最后，我们会的 在存在和不存在ANO1的情况下，量化睫状信号传导输出。这项工作将使我们能够阐明 ANO1在原发性纤毛生成中的作用，这是ANO1丧失的功能后果，并提供了洞察力 原发性纤毛离子通道生物学总体。了解分子途径将提供宝贵的见解 进入钙活化的氯化物通道和原发性纤毛中的离子通道的研究。这个基本 然后，在有机发育和纤毛病的背景下，知识可以应用于原发性纤毛 可以为治疗这些疾病的途径和目标提供新的见解。