Celsr1-mediated planar cell polarity: defining the adhesive interface and mechanisms of asymmetry

项目摘要 平面细胞极性(PCP)是一种核心的细胞接触和信号通路，在很大程度上是保守的 果蝇对哺乳动物来说，是早期发育和复杂的组织组织的基础， 多细胞生物。这项研究的长期目标是加深我们对中央PCP如何 蛋白质Celsr1介导了PCP建立和维持所需的粘附性相互作用。演技 与顶端-基底极性垂直，PCP是沿组织的细胞的集体和有组织的极化 飞机。五氯苯酚的一个显著例子是哺乳动物皮肤上体毛的有序排列。核心组件 该通路包括：Celsr(火烈鸟/FMI)、FrizzledFZ(FZ)、Vangl(Vangogh/Vang)、蓬乱(DVL)、皮刺 (PK)和迭戈(DGO)。PCP的一个显著特征是FZD/DV/DGO和Vangl/Pk at的不对称定位 通过CELSR的细胞间相互作用组织的连接复合体内的细胞边界，CELSR是一种大型非典型 钙粘附素是GPCR超家族的成员。干扰小鼠的CelsR功能会导致一系列 有害的表型包括神经管缺陷、异常的毛发模式和胚胎致死性。 此外，人类中的多个Celsr突变与神经管缺陷和诸如 如脊柱裂和心肌病。虽然CELSR在组织水平上的重要发育作用 通过对小鼠和苍蝇的研究，在分子和细胞水平上理解CelsR的功能 是缺乏的。为了阐明PCP不对称性是如何实现的，我们需要定义Celsr1的机制 细胞外黏附。小鼠皮肤是研究其保守机制的理想模型系统。 PCP和我们的实验室最近建立了一个自发极化的体外器官模型系统 测定PCP的不对称性和功能。结构照明等超分辨率成像技术 显微镜(SIM)和直接随机光学重建显微镜(DSTORM)进一步增强了我们的健壮性 研究表皮PCP的工具箱。这项提案的总体目标是调查PCP原则如何 蛋白质Celsr1介导细胞外黏附，以协调功能性PCP。我们假设Celsr1 钙粘附素重复序列介导不对称PCP蛋白所需的细胞间和侧向黏附相互作用 本土化和功能化。了解单个细胞-细胞在分子水平上的相互作用如何有助于 组织结构和功能将填补PCP领域的实质性和基础性知识空白。这 该方案综合了分子生物学、生物化学和先进的显微技术来阐明 五氯苯酚的不对称机制及其在体内外的作用。这些研究的完成将提供新的 从最基本的水平和水平深入了解哺乳动物表皮中PCP的调节机制 阐明我们对五氯苯酚复合体形成和功能的概念性理解。

Project Summary Planar cell polarity (PCP) is a core cell contact and signaling pathway that is largely conserved from Drosophila to mammals, and is fundamental to early development and tissue organization in complex, multicellular organisms. The long-term goal of this study is to further our understanding of how central PCP protein, Celsr1, mediates adhesive interactions required for the establishment and maintenance of PCP. Acting orthogonally to apico-basal polarity, PCP is the collective and organized polarization of cells along a tissue plane. A striking example of PCP is the ordered alignment of body hairs on mammalian skin. Core components of this pathway include: Celsr (Flamingo/Fmi), Frizzled (Fz), Vangl (VanGogh/Vang), Dishevelled (Dvl), Prickle (Pk) and Diego (Dgo). A hallmark feature of PCP is the asymmetric localization of Fzd/Dv/Dgo and Vangl/Pk at cell borders within a junctional complex organized via intercellular interactions of Celsr, a large atypical cadherin and member of the GPCR super-family. Disrupting Celsr function in mice leads to a range of detrimental phenotypes including neural tube defects, abnormal hair patterning and embryonic lethality. Further, multiple Celsr mutations in humans have been associated with neural tube defects and diseases such as spina bifida and cardiomyopathies. While important developmental roles for Celsr at the tissue-level have been established with mouse and fly studies, an understanding of Celsr function at the molecular and cell level is lacking. To elucidate how PCP asymmetry is achieved, we need to define the mechanisms of Celsr1 extracellular adhesion. The mouse skin is an ideal model system to investigate the conserved mechanisms of PCP and our lab has recently established a spontaneously polarizing in vitro, organotypic model system for assaying PCP asymmetry and function. Super-resolution imaging techniques such as structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) further add to our robust toolbox to study epidermal PCP. The overall goal of this proposal is to investigate how the principle PCP protein, Celsr1, mediates extracellular adhesion to coordinate functional PCP. We hypothesize that Celsr1 cadherin repeats mediate intercellular and lateral adhesive interactions required for asymmetrical PCP protein localization and function. Understanding how individual cell-cell interactions at the molecular level contribute to tissue structure and function will fill a substantial and fundamental knowledge gap in the PCP field. This proposal integrates molecular biology, biochemistry and advanced microscopy approaches to elucidate mechanisms of PCP asymmetry and function in vitro and in vivo. Completion of these studies will provide novel insight into the mechanisms the regulate PCP in the mammalian epidermis at the most fundamental level and illuminate our conceptual understanding of PCP complex formation and function.