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

项目总结 初级纤毛是一种类似于细胞触角的顶端细胞附属物，参与多种生理过程。 包括发育、增殖和细胞存活的过程。许多与纤毛相关的基因突变 蛋白质导致一系列被称为纤毛病的人类疾病，包括Joubert综合征和Bardet- 比德尔综合征。这些疾病表现出不同的临床表现，证明了初级纤毛功能的普遍存在。 影响人体许多器官系统的表型。作为基本的信号中枢，具有不同的蛋白质和 相对于细胞的其余部分，初级纤毛的形成和生长都受到高度的调节 功能。离子通道代表一类独特的蛋白质，它调节许多细胞中的信号事件 在纤毛方面，人们对纤毛的理解和理解才刚刚开始。我们使用的是 电生理和细胞成像相结合研究钙激活氯通道的作用 ANO1在调节原发纤毛发生和纤毛信号传导中的作用。我们最近发现，Ano1基因敲除会导致 与表达ANO1的细胞相比，纤毛更少、更短。此外，在小鼠中敲除Ano1会导致 组织畸形导致的新生儿死亡率反映了经典的纤毛病变特征。我们最近也是 描述了一种新的细胞结构，Nimbus，它含有ANO1，似乎具有时空相关性 纤毛发生。我们将验证ANO1在调节纤毛形成和功能方面的作用这一假设。 首先，我们将表征ANO1在纤毛发生过程中的定位和活性。当这些实验 在纤毛发生过程中，ANO1的活性起着重要作用，而纤毛发生的哪一步(S)对ANO1起着调节作用。最后，我们会 在ANO1存在和不存在的情况下量化纤毛信号输出。这项工作将使我们能够阐明 ANO1在原发纤毛发生中的作用，ANO1缺失的功能后果，并为深入了解 初级纤毛离子通道生物学总体。了解分子途径将提供有价值的见解 探讨初级纤毛中钙激活的氯离子通道和离子通道。这一基本原则 然后，知识可以应用于生物发育和纤毛疾病背景下的初级纤毛 可能为治疗这些疾病的途径和靶点提供新的见解。

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.