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

基本信息

批准号：
9910065
负责人：
Skylar Fisher
金额：
$ 4.55万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
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/9910065
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

项目摘要

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.

项目概要初级纤毛是一种类似于细胞天线的顶端细胞附属物，参与多种生理功能。包括发育、增殖和细胞存活等许多与睫状体相关的基因的突变。蛋白质会导致一系列称为纤毛病的人类疾病，其中包括朱伯特综合症和巴代综合症 Biedl 综合征证明了初级纤毛的普遍功能，这些疾病呈现出不同的临床症状。作为影响身体许多器官系统的表型，具有不同的蛋白质和相对于细胞的其余部分，初级纤毛的脂质成分在其形成和形成方面都受到高度调节。离子通道代表了一类独特的蛋白质，可调节许多细胞中的信号传导事件。过程，但在纤毛的背景下才刚刚开始被理解和欣赏。结合电生理学和细胞成像研究钙激活氯离子通道的作用 ANO1 调节初级纤毛发生和纤毛信号传导我们最近发现 Ano1 敲除会导致与表达 ANO1 的细胞相比，纤毛更少且更短。此外，敲除小鼠中的 Ano1 会导致我们最近还发现了由于组织畸形而导致的新生儿死亡，这反映了典型的纤毛病特征。表征了一种新颖的细胞结构，nimbus，它包含 ANO1 并且出现时空相关我们将检验 ANO1 在调节纤毛形成和功能中发挥作用的假设。首先，我们将描述 ANO1 在纤毛发生过程中的定位和活性。在纤毛发生过程中，ANO1 活性很重要，以及 ANO1 调节纤毛发生的哪些步骤。量化 ANO1 存在和不存在时的纤毛信号输出。这项工作将使我们能够阐明 ANO1 存在和不存在的情况。 ANO1 在初级纤毛发生中的作用、ANO1 缺失的功能后果，并提供对整体了解初级纤毛离子通道生物学将提供有价值的见解。进入初级纤毛中钙激活氯离子通道和离子通道的研究。然后可以在生物发育和纤毛病的背景下将知识应用于初级纤毛，可能为这些疾病的治疗途径和靶点提供新的见解。