Quantitative Single-Cell Analysis of Mammalian Notch Signaling States

描述（由申请人提供）：Notch信号通路使相邻细胞能够在不同过程（如血管生成、神经元分化和免疫系统发育）中协调发育命运。尽管Notch通路中的关键组分和相互作用是已知的，但仍不清楚它们如何共同作用以确定细胞的信号传导状态，其定义为使用特定Notch受体和配体发送和接收信号的定量能力。最近的研究表明，该系统的几个方面可以导致复杂的信号传导行为：首先，受体和配体以两种不同的方式相互作用，在同一细胞中相互抑制（顺式），同时在细胞之间有效地相互作用（反式）以发出信号。细胞发送或接收信号的能力很大程度上取决于这两种类型的相互作用。其次，哺乳动物具有多种类型的受体和配体，它们以不同的强度相互作用，并且在自然系统中经常共表达。第三，三种哺乳动物边缘蛋白可以以不同的和配体特异性的方式改变受体-配体相互作用强度。因此，细胞可以表现出非直观的信号状态，即使具有相对较少的组件。为了了解在自然过程中发生了什么样的信号传导状态，以及它们能够实现什么类型的信号传导行为，该提案旨在对Notch信号传导状态如何由受体，配体和边缘蛋白的表达水平决定进行定量和预测性理解。为此，我们将构建一组细胞系，其允许控制配体和Fringe表达水平，并读出所得Notch活性。我们将对这些细胞系进行一项测定，该测定将定量评估单个细胞表面上同时可用的Notch配体和受体的水平。我们将使用延时显微镜和定量图像分析系统地测量在单个细胞水平上不同配体-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Fringe表达水平和类型的依赖性，以及多种Fringe蛋白之间相互作用的影响。我们将开发一个数学建模框架，使用这些数据从组分表达水平预测单个细胞的信号状态。我们将使用微孔图案化系统来测试这些预测，该系统允许我们通过将单个细胞与单个邻居共培养来分析单个细胞的发送和接收状态。这些方法将允许 我们重建和分析了一组不同的Notch信号配置自下而上，并提供了一个全面的看法，这一关键信号通路的信号库。这些结果将为许多哺乳动物的发育系统提供深入了解，并有助于对Notch依赖性疾病过程进行合理干预。 公共卫生相关性：通过Notch途径的细胞间信号传导介导关键和多样的发育模式化过程，包括神经发生、体节发生、血管生成和造血发育。Notch信号传导的破坏与许多疾病有关。该提案将提供对Notch通路的组成部分如何共同决定单个细胞的信号状态的理解，以及通路组成部分的扰动如何改变这些信号状态，例如疾病中发生的那些。该方法包括定量单细胞分析的重组信号通路与数学建模。这些结果将阐明Notch功能的机制，并支持药物干预的发展。

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. PUBLIC HEALTH RELEVANCE: Intercellular signaling through the Notch pathway mediates critical and diverse developmental patterning processes including neurogenesis, somitogenesis, angiogenesis, and hematopoietic development. The disruption of Notch signaling is implicated in a number of diseases. This proposal will provide an understanding of how components of the Notch pathway together determine the signaling states of individual cells, and how perturbations of pathway components, such as those that occur in disease, alter these signaling states. The approach involves quantitative single-cell analysis of reconstituted signaling pathways together with mathematical modeling. The results will elucidate mechanisms of Notch function and support the development of pharmacological interventions.