Dynamics of Notch Signaling

项目摘要 信号转导通路是生命的基础，往往是疾病治疗的关键靶点。 然而，准确地理解细胞内的动力学、动力学和化学计量的信号是如何 传递的知识对于全面、更准确地了解信令流程至关重要- 由于实时观察信令事件的技术障碍，许多路径都无法实现 时间，单位为单元格。技术方面的最新进展，包括邻近标记方法和光片 现在终于提出了解决方案，将我们对体内信号传递的支离破碎的观点结合在一起。建房 论Blacklow实验室在Notch信号机制方面的专业知识与Kirchhausen的专业知识 在高级显微镜实验室，我们建议使用Notch信号系统作为模型来精确定义 Notch信号在正常和致病状态下激活所需的一系列事件，并在这样做的过程中， 可广泛应用于其他途径和系统的方法和计算可视化工具。 Notch信号是这项工作的理想模型信号系统，因为它对细胞产生关键影响 所有后生动物的分化及其失调与多种疾病有关，包括 许多人类癌症的发病机制。此外，该信令机制的基本方面，包括 配体和受体分子的动力学，激活部位的配体-受体复合体的化学计量比， 激活Notch蛋白降解的时机和位置，以及Notch从质膜到细胞核的路径， 仍然没有完全被理解。在这里，我们将通过使用APEX近邻来解决这些知识差距 标记结合定量质谱学研究Notch的传递途径 细胞内结构域从质膜到细胞核，并通过实施晶格光片显微镜来 可视化Notch信号转导的分子事件。在前期工作中，我们使用了CRISPR/CAS9 在SVG-A永生化星形胶质细胞中进行基因组标记以创建Notch-APEX2融合蛋白 标记，我们对试点实验的初步分析揭示了蛋白质标记的动态变化 在不同的细胞隔间中作为时间的函数，证实了该方法的可行性。我们还有 为LLSM设计荧光标记的受体和配体敲打蛋白。现在我们将使用这种方法 为了量化在细胞-细胞接触部位聚集的受体和配体分子的数量 时间的作用，并确定每个基因必须存在多少个拷贝才能诱导受体切割和 激活靶基因表达。成功完成这些目标将提供前所未有的决心， 在活细胞中生理Notch信号转导所需的基本事件的空间和时间。 我们希望不仅回答长期存在的关于Notch激活的分子事件的问题 但也为机械分析开辟了一个全新的生物学领域。

Project Summary Signal transduction pathways underlie the very basis of life and are often critical targets for disease therapeutics. Yet, precise understanding of the intracellular dynamics, kinetics, and stoichiometry of how signals are transmitted – knowledge that is critical to capturing a holistic and more accurate view of signaling processes – has not been attainable for many pathways due to technological barriers to observing signaling events in real time in cells. Recent advances in technology, including proximity labeling approaches and light sheet microscopy, are finally now presenting solutions to knit together our fragmented view of signaling in vivo. Building on the expertise of the Blacklow laboratory in Notch signaling mechanism with the expertise of the Kirchhausen laboratory in advanced microscopy, we propose to use the Notch signaling system as a model to define precisely the series of events required for Notch signal activation in normal and pathogenic states, and in so doing, develop approaches and computational visualization tools that can be broadly applied to other pathways and systems. Notch signaling is an ideal model signaling system for this work because it exerts a critical influence on cell differentiation in all metazoans and its misregulation is associated with diverse diseases, including the pathogenesis of many human cancers. Moreover, fundamental facets of this signaling mechanism, including the dynamics of ligand and receptor molecules, the stoichiometry of ligand-receptor complexes at sites of activation, the timing and location of activating Notch proteolysis, and the path of Notch from plasma membrane to nucleus, remain incompletely understood. Here, we will address these gaps in knowledge by using APEX proximity labeling coupled with quantitative mass spectrometry to elucidate the pathway for passage of the Notch intracellular domain from plasma membrane to nucleus, and by implementing lattice light sheet microscopy to visualize the molecular events of Notch signal transduction. In preliminary work, we have used CRISPR/Cas9 genomic labeling in SVG-A immortalized astrocytes to create a Notch-APEX2 fusion protein for proximity labeling, and our preliminary analysis of a pilot experiment reveals dynamic changes in the labeling of proteins in different cellular compartments as a function of time, confirming the feasibility of this approach. We have also engineered fluorescently labeled receptor and ligand knockin proteins for LLSM. Now we will use this approach to quantify the number of receptor and ligand molecules that come together at the site of cell-cell contact as a function of time, and determine how many copies of each must be present to induce receptor cleavage and activate target gene expression. Successful completion of these aims will provide unprecedented resolution, in both space and time, of the fundamental events required for physiologic Notch signal transduction in living cells. We expect to not only answer long-standing questions about the molecular events involved in activation of Notch signals but to also open up a whole new realm of biology to mechanistic analysis.