Proj 1: Imaging Notch Interations with Members of Its Pathway

Notch信号是后生动物用来指导细胞命运的进化保守机制 决定，增殖和凋亡在所有发展阶段，包括自我更新的成年组织。 异常Notch信号传导与癌症有关，特别是在癌症干细胞的新兴领域。 哺乳动物细胞含有四种Notch受体和五种Delta和Jagged同源配体; 本研究旨在阐明背景对单个Notch受体亚型介导信号的重要性 使用非侵入性分子成像策略。我们建议开发使能技术， 有助于高通量筛选能够调节Notch的试剂和基因产物 癌症、遗传性疾病和促进组织工程的信号通路。全面 对如何以依赖于上下文的方式操纵单个受体的机械理解仍然是 逃避调查人员。我们建议开发一个真实的时间成像系统，将超越目前的 可用报表提供真实的时间，量化核算单个Notch的激活状态 受体亚型我们正在开发的报告系统是基于荧光素酶互补 在华盛顿大学医学院的ICMIC分子报告核心中开发的成像技术 医学和可视化之间的相互作用的特定Notch细胞内结构域（NICD） 和共同的核辅因子RBPjk。该系统是通用的，它可以适用于研究路径 在不同的细胞类型，并可以很容易地修改，以监测与其他癌症相关的合作伙伴的相互作用 例如NF-kB途径的组分。虽然成像技术正在开发中， 应用不直接适用于患者的成像，因为它需要蛋白质工程，这些 这些方法将有助于开发和评估Notch相关疾病的新疗法。

Notch signaling is an evolutionary conserved mechanism used by metazoans to direct cell fate decisions, proliferation and apoptosis at all stages of development, including self-renewing adult tissues. Aberrant Notch signaling is implicated in cancer, especially in the emerging field of cancer stem cells. Mammalian cells contain four Notch receptors and five Delta and Jagged cognate ligands; the overall goal of this proposal is to elucidate the significance of context on individual Notch receptor subtype-mediated signals using non-invasive molecular imaging strategies. We propose to develop enabling technologies that will facilitate high throughput screening for agents and gene products that are capable of modulating the Notch signaling pathway in cancer, inherited diseases and facilitate tissue engineering. A comprehensive mechanistic understanding of how to manipulate individual receptors in a context-dependent manner still eludes investigators. We propose to develop a real time imaging system that will go beyond the currently available reporters in providing real time, quantitative accounting of the activation status of individual Notch receptor subtypes. The reporter system we are developing is based on the luciferase complementation imaging technology developed in the ICMIC Molecular Reporter Core at Washington University School of Medicine and enables visualization of the interactions between a specific Notch intracellular domain (NICD) and the common nuclear cofactor RBPjk. The system is versatile; it can be adaptedfor studying the pathway in different cell types and can be easily modified to monitor interaction with other cancer-relevant partners such as components of the NF-kB pathway. While the imaging technology being developed in this application is not directly applicable to imaging in patients as it requires protein engineering, these methodologies will help develop and evaluate novel therapies for Notch-related diseases.