Role of Clic in Epithelial Morphogenesis

描述(由申请人提供)：本申请的长期目标是阐明最近描述的被称为CLICS(氯离子细胞内通道)的蛋白质家族在上皮形态发生中的作用。虽然首字母缩写“CLIC”暗示这些蛋白质可以作为离子通道发挥作用，但新出现的证据表明，它们也具有不同于离子运输的替代功能。需要检验的中心假设是，CLICS是在上皮细胞形态发生过程中控制细胞骨架活动和极性的调节蛋白。通过遗传学、生化和亚细胞定位研究，已经确定了与肌动蛋白细胞骨架和信号转导相关的基因；然而，它们的确切作用仍有待确定。最近的研究表明，clic基因的破坏会导致线虫的管状囊形成和小鼠感觉上皮细胞的结构性退化而导致耳聋。此外，人类基因已被认为是肿瘤形成和转移的诊断指标和致病因素。老生常谈的功能还没有经过严格的测试。在这项提案中，果蝇将被用作一个模型系统，以揭示CLICS的功能意义和细胞角色。与脊椎动物不同，脊椎动物有多个clic基因和剪接变体，而果蝇只有一个clic基因。我们的初步数据表明，果蝇Clic在功能上与果蝇发育所必需的细胞骨架蛋白和信号分子相互作用。本研究的主要目的是研究CLIC在胚胎上皮细胞形态发生过程中的细胞骨架调控作用，并拓宽我们对其在胚胎发育信号转导中作用的认识：1.探讨CLIC在胚胎上皮细胞形态发生中的作用。我们将测试果蝇在早期胚胎发育中是否需要确定分割决定因素的位置。此外，我们还将研究CCIC在胚胎发育后期上皮细胞内陷和上皮片运动中的作用。CLIC和已知的调节蛋白之间的潜在相互作用对上皮的极性至关重要，将从遗传和生化方面进行测试。2.鉴定在胚胎发育过程中与CLIC相互作用的基因。我们将确定CLIC的遗传修饰物，验证相互作用，并评估CLIC修饰物在胚胎发育过程中的细胞功能。此外，我们还将测试在上皮片形态发生运动过程中与CLIC发生重要功能相互作用的候选调控基因。由于许多蛋白质在脊椎动物和无脊椎动物物种中功能保守，因此对果蝇ClyC功能的分析很可能有助于更好地了解ClyC在人类中的功能。 公共卫生评论：这项提案的主要目标是确定在人类和昆虫之间高度保守的CLIC蛋白是如何控制细胞的形状和运动的。CLIC蛋白与人类健康相关，因为它们对听力和平衡是必不可少的，并与肿瘤的形成和转移有关。果蝇将被用作一个模型系统，以了解CLIC如何与其他已知在胚胎发育过程中对细胞形状和运动具有重要作用的基因和蛋白质一起工作。

DESCRIPTION (provided by applicant): The long-term goal of this application is to elucidate the role of a recently described family of proteins termed Clics (chloride intracellular channels) in epithelial morphogenesis. Although the acronym "Clic" implies that these proteins may function as ion channels, emerging evidence indicates that they also have alternative functions distinct from ion transport. The central hypothesis to be tested is that Clics are regulatory proteins that control cytoskeletal activity and polarity during epithelial cell morphogenesis. Clics have been identified in association with the actin cytoskeleton and signal transduction through genetic, biochemical, and subcellular localization studies; however, their precise roles remain to be defined. Recent studies have shown that disruption of Clic genes causes tubular cyst formation in C. elegans and deafness due to structural degeneration of sensory epithelial cells in mice. In addition, human Clics have been implicated as diagnostic indicators and causative factors in tumor formation and metastasis. The function of Clics has not been rigorously tested. In this proposal, Drosophila will be used as a model system to unravel the functional significance and cellular roles of Clics. Unlike vertebrates, which have multiple Clic genes and splice variants, Drosophila has only one Clic gene. Our preliminary data indicate that Drosophila Clic functionally interacts with cytoskeletal proteins and signaling molecules essential to fly development. The major objectives are to examine the role of Clic in cytoskeletal regulation during embryonic epithelial morphogenesis and to broaden our perspective on the role of Clic in signaling during embryonic development as follows: 1. Investigate the role of Clic during embryonic epithelial morphogenesis. We will test whether Drosophila Clic is required for establishing the position of segmentation determinants in early embryogenesis. In addition, we will investigate the role of Clic in epithelial invagination and epithelial sheet movement late in embryogenesis. Potential interactions between Clic and known regulatory proteins key to epithelial polarity will be tested genetically and biochemically. 2. Identify genes which interact with Clic during embryonic development. We will identify genetic modifiers of Clic, verify the interactions, and assess the cellular functions of Clic modifiers during embryonic development. In addition, we will test candidate regulatory genes for functionally important interactions with Clic during morphogenetic movements of epithelial sheets. Since many proteins are functionally conserved among vertebrate and invertebrate species, it is likely that analysis of Clic function in Drosophila will lead to a better understanding of Clic function in humans. PUBLIC HEALTH REVELANCE: The main goal of this proposal is to determine how Clic proteins, which are highly conserved between humans and insects, control cell shape and movement. Clic proteins are relevant to human health because they are essential for hearing and balance and have been implicated in tumor formation and metastasis. The fruit fly will be used as a model system to understand how Clic works with other genes and proteins known to be important for cell shape and movement during embryonic development.