Analysis of fringe-dependent cell-signaling

描述（由申请人提供）：在本提案中，我们调查了 调节Notch信号通路的机制和意义 边缘。Notch信号具有重要的生物学和医学意义，因为 大多数动物组织似乎至少在某些方面利用Notch信号传导 他们的发展。人类疾病的范围已经与 迄今为止，Notch途径中的突变反映了Notch的多样性。 在人类生物学中的功能：异常的Notch信号传导与 白血病，一种与中风和痴呆有关的先天性综合征， 与肝脏、心血管、骨骼和其他相关的先天性综合征 缺陷和与中轴骨缺陷相关的先天性综合征。 Fringe作为Notch信号传导途径的调节剂发挥作用，并编码一种 延长O-连接岩藻糖的新型糖基转移酶。概述的项目 在这个更新应用程序是针对了解如何 Fringe依赖性碳水化合物修饰影响Notch激活和 其他细胞过程。为了阐明迷离档案调节 Notch激活，我们将确定蛋白质，和EGF模块内 这些蛋白质，是边缘的生物相关目标， 改变Notch，Serrate， 和Delta，并测定这些突变蛋白在果蝇中的活性。我们 还将分析不同的碳水化合物结构对Notch的作用， 分析多肽的表达和体内需求 岩藻糖基转移酶和B-半乳糖苷唾液酸转移酶。为了定义 Fringe调节Notch信号的分子机制，我们将 建立体外Notch-配体结合测定。然后我们将使用这个分析 与特定EGF结构域的改变及其 碳水化合物结构对体内Notch激活的物理作用 Notch及其配体之间的相互作用。Notch激活的定位 Fringe将果蝇的翅膀分为背翅和腹翅 隔间我们将检验我们的假设，即有丝分裂平行于 隔室边界直接可视化有丝分裂纺锤体。然后我们将 评估的作用和调控定向有丝分裂在翅膀通过分析 组织极性通路和Notch通路的成员对 这个过程这些研究将促进我们对监管的理解 和Notch信号通路的功能，并将提供第一个 差异受体糖基化如何影响 受体-配体相互作用

DESCRIPTION (provided by applicant): In this proposal, we investigate the mechanisms and implications of the regulation of the Notch signaling pathway by fringe. Notch signaling is of great biological and medical importance, because most animal tissues appear to utilize Notch signaling for at least some aspects of their development. The range of human diseases that have been associated with mutations in the Notch pathway thus far reflects the diversity of Notch functions in human biology: aberrant Notch signaling has been linked to leukemia, a congenital syndrome associated with stroke and dementia, a congenital syndrome associated with liver, cardiovascular, skeletal and other defects, and a congenital syndrome associated with axial skeletal defects. Fringe functions as a modulator of the Notch signaling pathway and encodes a novel glycosyltransferase that elongates 0-linked fucose. The projects outlined in this renewal application are directed towards understanding how Fringe-dependent carbohydrate modifications influence Notch activation and other cellular processes. To elucidate the mechanisms by which Fringe modulates Notch activation, we will identify the proteins, and the EGF modules within those proteins, that are the biologically relevant targets of Fringe by altering consensus sites for addition of 0-linked fucose within Notch, Serrate, and Delta, and assaying the activity of these mutant proteins in Drosophila. We will also analyze the role of distinct carbohydrate structures on Notch by analyzing the expression and in vivo requirements for a polypeptide fucosyltransferase and a B-galactoside sialyltransferase. In order to define the molecular mechanism by which Fringe modulates Notch signaling, we will establish an in vitro Notch-ligand binding assay. We will then use this assay to relate the influence of alterations in specific EGF domains and their carbohydrate structures on Notch activation in vivo to the physical interactions between Notch and its ligands. The positioning of Notch activation by Fringe separates the Drosophila wing into distinct dorsal and ventral compartments. We will test our hypothesis that mitoses are oriented parallel to the compartment border by directly visualizing mitotic spindles. We will then assess the role and regulation of oriented mitoses in the wing by analyzing the influence of members of the tissue polarity pathway and the Notch pathway on this process. These studies will advance our understanding of the regulation and functions of the Notch signaling pathway, and will provide the first detailed picture of how differential receptor glycosylation can influence receptor-ligand interactions.