Control of Notch signaling by endocytosis

描述（由申请人提供）：Notch信号传导是所有后生动物中每个组织发育所普遍需要的，并且该机制的缺陷与许多人类发育疾病和癌症相关。Notch途径的一个独特特征是其激活和调节依赖于内吞作用的程度。Notch信号传导的一个神秘特征是配体必须被信号传导细胞内化，以便它们激活相邻细胞上的Notch受体。我们提出的研究旨在确定为什么配体内吞作用是必要的信号。此外，我们探讨如何调节内吞因子有助于调节信号。两种内吞蛋白，Epsin和Epilin，对于配体内化和信号传导是绝对必要的。我们建议调查如何Epsin和Escherichilin在信号细胞中的功能，以及它们是如何被调节的。特别是，调控的Epsin活性的泛素化将被探索。此外，我们建议确定其他内吞蛋白和调节所需的信号细胞。我们使用的方法包括果蝇遗传学、发育中眼睛的免疫组织化学和生物化学。首先，我们将确定哪些Epsin蛋白相互作用模块是Delta信号传导所必需的。我们将产生转基因果蝇，表达各种突变的Epsin蛋白，并确定哪些突变体支持信号传导，Delta内吞作用，Epsin泛素化，质膜定位和正常水平的Epsin积累。这些实验将解决有关Epsin功能和调节的争议问题。其次，我们测试两个假设，为什么需要的信号。在胞吞作用中，β-内酰胺酶具有不同的作用，如果我们能够确定哪个作用对信号传导很重要，我们就能够理解为什么δ-内酰胺酶是必需的。该方法是为了测试不同转基因在果蝇中的表达是否会抑制信号传导中对白藜芦醇的需求。此外，我们进行了筛选与生长素相互作用的基因。第三，我们建议研究泛素调节Epsin的机制。Epsin通过泛素化失活，而Fat facets的去泛素化激活Epsin。我们的目标是了解这个泛素化循环的Delta信号的相关性。我们建议确定泛素连接酶，泛素化Epsin，分析缺乏连接酶的苍蝇的表型，并使用连接酶的生化实验，以确定模式的Epsin泛素化。此外，我们建议使用质谱法来绘制从苍蝇中纯化的Epsin的泛素化位点，并确定是否存在泛素链，如果存在，它们是如何连接的。第四，我们提出了遗传和生物化学实验来检验这一假设，即Ral GT3通过抑制Epsin的水平来负调节Delta信号传导。第五，我们计划描述我们在筛选与Epsin基因相互作用的基因时发现的9个基因，液体小面。我们认为其中一些基因可能编码Delta信号的调节因子，这些调节因子通过Epsin发挥作用。公共卫生相关性：Notch信号通路在后生动物中普遍用于控制每种细胞类型的细胞增殖、特化、分化和生长。因此，Notch信号传导的缺陷与多种人类发育疾病和癌症相关。因此，理解Notch通路的调节是至关重要的，果蝇遗传学和生物化学的结合是研究这些过程的有力方式。

DESCRIPTION (provided by applicant): Notch signaling is required universally for development in every tissue in all metazoans, and defects in the mechanism are associated with many human developmental diseases and cancer. A peculiar feature of the Notch pathway is the extent to which its activation and regulation depend on endocytosis. A mysterious feature of Notch signaling is that ligand must be internalized by the signaling cells in order for them to activate the Notch receptor on adjacent cells. The research we propose seeks to determine why ligand endocytosis is necessary for signaling. In addition, we explore how regulation of endocytic factors contributes to regulation of signaling. Two endocytic proteins, Epsin and Auxilin, are absolutely necessary for ligand internalization and signaling. We propose to investigate how Epsin and Auxilin function in the signaling cells, and how they are regulated. In particular, regulation of Epsin activity by ubiquitination will be explored. Moreover, we propose to identify other endocytic proteins and regulators required in the signaling cells. The methodology we use includes Drosophila genetics, immunohistochemistry of developing eyes, and biochemistry. First, we will determine which protein interaction modules of Epsin are required for Delta signaling. We will generate transgenic flies that express a variety of mutant Epsin proteins, and determine which mutants support signaling, Delta endocytosis, Epsin ubiquitination, plasma membrane localization, and normal levels of Epsin accumulation. These experiments will resolve controversial issues regarding the function and regulation of Epsin. Second, we test two hypotheses as to why Auxilin is required for signaling. Auxilin has diverse roles in endocytosis, and if we can determine which role is important for signaling, we may be able to understand why Delta endocytosis is necessary. The approach is to test if expression of different transgenes in flies will obviate the requirement for Auxilin in signaling. In addition, we perform a screen for genes that interact with auxilin. Third, we propose to investigate the mechanism by which Ubiquitin regulates Epsin. Epsin is inactivated by ubiquitination, and deubiquitination by Fat facets activates Epsin. We aim to understand the relevance to Delta signaling of this ubiquitination cycle. We propose to identify the Ubiquitin-ligase that ubiquitinates Epsin, to analyze the phenotypes of flies that lack the ligase, and to use the ligase in biochemical experiments to determine the mode of Epsin ubiquitination. In addition, we propose to use mass spectrometry to map the sites of ubiquitination on Epsin purified from flies, and to determine whether Ubiquitin chains are present, and if so, how they are linked. Fourth, we propose genetic and biochemical experiments to test the hypothesis that the Ral GTPase negatively regulates Delta signaling by depressing the levels of Epsin. Fifth, we plan to characterize nine genes that we identified in a screen for genes that interact with the Epsin gene, liquid facets. We think that some of these genes are likely to encode regulators of Delta signaling that function through Epsin. PUBLIC HEALTH RELEVANCE: The Notch signaling pathway is used universally in metazoans to control cell proliferation, specification, differentiation, and growth of every cell type. Defects in Notch signaling are thus associated with a wide variety of human developmental diseases and cancer. For this reason, an understanding of Notch pathway regulation is critically important, and the combination of Drosophila genetics and biochemistry is a power manner in which to study these processes.