Quantitative Single-Cell Analysis of Mammalian Notch Signaling States

描述(由申请人提供)：Notch信号通路使邻近细胞能够在不同的过程中协调发育命运，如血管生成、神经元分化和免疫系统发育。虽然Notch途径中的关键成分和相互作用是已知的，但它们如何协同工作来确定细胞的信号状态仍不清楚，信号状态的定义是细胞使用特定的Notch受体和配体发送和接收信号的定量能力。最近的工作表明，该系统的几个方面可以导致复杂的信号行为：首先，受体和配体以两种不同的方式相互作用，在同一细胞内(顺式)相互抑制，同时在细胞之间(反式)有效地相互作用以产生信号。细胞发送或接收信号的能力很大程度上取决于两种类型的相互作用。其次，哺乳动物有多种类型的受体和配体，它们相互作用的强度不同，并且经常在自然系统中共表达。第三，这三种哺乳动物边缘蛋白可以以不同的和特定于配体的方式改变受体-配体的相互作用强度。因此，即使在组件相对较少的情况下，细胞也可能表现出非直观的信号状态。为了了解在自然过程中发生什么信号状态，以及它们启动了什么类型的信号行为，本提案试图对Notch信号状态是如何由受体、配体和边缘蛋白的表达水平决定的进行定量和预测性的理解。为此，我们将构建一组细胞系，允许控制配体和条纹表达水平，并读出由此产生的Notch活性。我们将对这些细胞系进行检测，以定量评估单个细胞表面同时存在的Notch配体和受体的水平。我们将使用时间推移显微镜和定量图像分析，在单个细胞水平上系统地测量不同配体-受体组合之间的顺式和反式相互作用强度。我们将进一步分析这些相互作用对Fringer表达水平和类型的依赖性，以及多个Frage蛋白之间相互作用的影响。我们将开发一个数学建模框架，使用这些数据从成分表达水平预测单个细胞的信号状态。我们将使用微孔模式系统来测试这些预测，该系统允许我们通过与单个邻居共同培养单个细胞来分析其发送和接收状态。这些方法将允许 我们将自下而上地重组和分析一组不同的Notch信令配置，并提供这一关键信令途径的信令曲目的全面视图。这一结果将提供对众多哺乳动物发育系统的洞察，并可能促进对Notch依赖疾病过程的理性干预。

DESCRIPTION (provided by applicant): The Notch signaling pathway enables neighboring cells to coordinate developmental fates in diverse processes such as angiogenesis, neuronal differentiation, and immune system development. Although key components and interactions in the Notch pathway are known, it remains unclear how they work together to determine the cell's signaling state, defined as its quantitative ability to send and receive signals using particular Notch receptors and ligands. Recent work suggests that several aspects of the system can lead to complex signaling behaviors: First, receptors and ligands interact in two distinct ways, inhibiting each other in the same cell (in cis) while productively interacting between cells (in trans) to signal. The ability of a cell to send or receive signals depends strongly on both types o interactions. Second, mammals have multiple types of receptors and ligands, which interact with different strengths, and are frequently co-expressed in natural systems. Third, the three mammalian Fringe proteins can modify receptor-ligand interaction strengths in distinct and ligand-specific ways. Consequently, cells can exhibit non-intuitive signaling states even with relatively few components. In order to understand what signaling states occur in natural processes, and what types of signaling behaviors they enable, this proposal seeks to develop a quantitative and predictive understanding of how the Notch signaling state is determined by the expression levels of receptors, ligands, and Fringe proteins. To do so, we will construct a set of cell lines that allow control of ligand and Fringe expression level, and readout of the resulting Notch activity. We will subject these cell lines to an assay that will quantitatively assess the levels of Notch ligands and receptors simultaneously available on the surface of individual cells. We will use time-lapse microscopy and quantitative image analysis to systematically measure both cis and trans interaction strengths between different ligand-receptor combinations at the level of individual cells. We will further analyze the dependence of these interactions on the level and type of Fringe expression, as well as the effects of interactions between multiple Fringe proteins. We will develop a mathematical modeling framework that uses these data to predict the signaling states of individual cells from component expression levels. We will test these predictions using a microwell patterning system that allows us to analyze both send and receive states of a single cell by co-culturing it with a single neighbor. These methods will allow us to reconstitute and analyze a diverse set of Notch signaling configurations from the bottom up, and provide a comprehensive view of the signaling repertoire of this critical signaling pathway. The results will provide insight into numerous mammalian developmental systems, and could facilitate rational intervention into Notch-dependent disease processes.