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

项目摘要 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。与纤毛相关的许多基因的突变 蛋白质引起称为纤毛病的人类疾病家族，包括Joubert综合征和Bardet综合征， 比德尔综合征。这些疾病的临床表现多种多样，证明了初级纤毛的普遍功能。 影响身体许多器官系统的表型。作为具有不同蛋白质的基本信号中枢， 相对于细胞的其余部分，初级纤毛的脂质组成在它们的形成和 功能离子通道是一类独特的蛋白质，在许多细胞中调节信号事件。 过程，但只是开始被理解和赞赏的上下文中的纤毛。我们使用的是 结合电生理学和细胞成像来研究钙激活氯通道的作用 ANO1在调节初级纤毛发生和纤毛信号传导中的作用。我们最近发现Ano1敲除导致 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导致 由于组织畸形导致的新生儿死亡反映了典型的纤毛病变特征。我们最近还 其特征在于一种新的细胞结构，即Nimbus，它含有ANO1，并且与时空相关 到纤毛发生我们将检验ANO1在调节纤毛形成和功能中起作用的假设。 首先，我们将描述的本地化和活性的ANO1在纤毛发生。这些实验当 在纤毛发生期间，ANO1活性是重要的，以及ANO1调节纤毛发生的步骤。最后我们将 在存在和不存在ANO1的情况下定量纤毛信号传导输出。这项工作将使我们能够阐明 ANO1在初级纤毛发生中的作用，ANO1缺失的功能后果，并提供了对 初级纤毛离子通道生物学整体。了解分子途径将提供有价值的见解 研究初级纤毛中的钙激活氯离子通道和离子通道。这一根本 然后，知识可以应用于生物体发育和纤毛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.