Ubiquitin-like protein modifications in planar cell polarity

描述(申请人提供)：上皮细胞的极化在两个轴上是明显的，在普遍存在的根尖-基底外侧轴和作为第二轴的上皮平面内，后者通常被称为平面细胞极性(或PCP)。PCP的例子存在于几乎所有的器官中，例如，在哺乳动物中，最明显的是皮肤发育方面的毛囊取向或内部器官的细胞排列，如内耳上皮及其感觉纤毛。在果蝇中，所有成体的角质结构和器官都显示 PCP的显著特征使果蝇PCP的建立研究成为研究发育模式和疾病过程的典范。对果蝇的分析已经建立了一条围绕FrizzledFz/PCP核心因子盒和相关调控因子的保守分子途径。这个核心的FZ/PCP途径及其调控成分在整个进化过程中是保守的，不仅在上皮器官中调节细胞极化的许多方面，而且在哺乳动物中也调节细胞定向迁移的许多方面 原肠形成和神经形成过程中间充质细胞的数量。尽管核心FZ/PCP因子之间相互作用的框架正在形成，但人们对它们相互作用的实际分子机制知之甚少。这项应用的范围是从基因组范围的筛查中跟踪非常有趣的基因鉴定，以寻找核心PCP因子的新调节因子。基于有趣的初步数据，我们提出的具体目标是：(1)研究后期促进复合体的泛素连接酶活性(APC/C)的作用，并确定该复合体中的哪些成分在PCP中起作用；(2)定义由CG15283编码的新型泛素样修饰物在PCP建立中的功能，以及它如何在PCP信号转导过程中调节核心FZ/PCP因子或APC/C组分的活性。以及(3)鉴定APC/C的PCP特异性底物，并通过APC/C定义它们的分子调控。我们已经建立了几种方法，使我们能够通过结合果蝇体内功能研究、生化实验和细胞培养分析来实现这些目标。由于APC/C在细胞周期调控外的作用和保守的CG15283肽的功能分别很大程度上是未知的或完全未知的，我们的应用将为我们(S)提供第一次深入了解这些机制的机会(S)。FZ/PCP的建立与几种医学异常有关，包括耳聋、癌症、多囊肾病和纤毛疾病。因此，在这一应用中获得的信息不仅将促进我们对PCP调节和器官模式的理解，而且在几种疾病背景下也将具有医学意义。

DESCRIPTION (provided by applicant): Polarization of epithelial cells is evident in two axes, in the ubiquitous apical-basolateral axis and within the plane of the epithelium as a second axis, the latter generally referred to as Planar Cell Polarity (or PCP). Examples of PCP are present in almost all organs, and in mammals, for example, most obvious in aspects of skin development with hair follicle orientation or cellular arrangements in internal organs, like the inner ear epithelium with its sensory cilia. In Drosophila, all adult cuticular structures and organs display striking PCP features, which makes the study of PCP establishment in Drosophila serve as a paradigm for the process in developmental patterning and disease in general. Analyses in Drosophila have established a conserved molecular pathway anchored around the Frizzled (Fz)/PCP core factor cassette and associated regulatory factors. This core Fz/PCP pathway and its regulatory components are conserved throughout evolution regulating many aspects of cellular polarization not only in epithelial organs, but in mammals also in directed cell migration of mesenchymal cells during gastrulation and neurulation. Although a framework of the interactions among the core Fz/PCP factors is emerging, little is known about the actual molecular mechanisms underlying their interactions. The scope of this application is to follow-up on very interesting gene identifications from a genome wide screen for novel regulators of the core PCP factors. Based on interesting preliminary data, we propose as Specific Aims to (1) address the role of the Ubiquitin-ligase activity of the Anaphase Promoting Complex (APC/C) and define which components of this complex act in PCP and how, (2) define the function of the novel ubiquitin-like modifier encoded by CG15283 in PCP establishment and how it might regulate the activity of core Fz/PCP factors or APC/C components during PCP signaling, and (3) identify the PCP specific substrates of the APC/C and define their molecular regulation by the APC/C. We have established several assays that will allow us to address these aims via a combination of functional in vivo studies in Drosophila, biochemical experiments, and cell culture analyses. As the roles of the APC/C outside cell cycle control and a function of the conserved CG15283 peptide are largely obscure or completely unknown, respectively, our application will provide first insight(s) into the mechanism(s) of these. Fz/PCP establishment has been linked to several medical abnormalities, including deafness, cancer, polycystic kidney disease, and ciliopathies. Thus the information acquired in this application will both advance our understanding of PCP regulation and organ patterning, and will also be of medical relevance in several disease contexts.