Quantitative Single-Cell Analysis of Mammalian Notch Signaling States

哺乳动物 Notch 信号状态的定量单细胞分析

• 批准号: 8440168
• 负责人: MICHAEL B ELOWITZ
• 金额: $ 34.24万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-26 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440168
• 关键词: Address; Behavior; Binding; Biochemical; Biological Assay; Biological Process; Cell Culture Techniques; Cell Line; Cells; Cellular Structures; Coculture Techniques; Communication; Complex; Computer Simulation; Data; Dependence; Development; Disease; Engineering; Exhibits; Genetic; Hematopoietic; Image Analysis; Immune system; Individual; Intervention; Knowledge; Lead; Ligands; Mammals; Manic; Measurement; Measures; Mediating; Methods; Microscopy; Modeling; Neuronal Differentiation; Noise; Notch Signaling Pathway; Organism; Pathway interactions; Pattern; Process; Quantitative Microscopy; Reading; Role; Signal Pathway; Signal Transduction; Specificity; Surface; System; Systems Development; Testing; Time; To specify; Work; angiogenesis; base; combinatorial; fringe protein; glycosyltransferase; insight; mathematical model; movie; neurogenesis; notch protein; receptor; receptor expression; reconstitution; single cell analysis; somitogenesis; time use

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.

描述（由申请人提供）：Notch信号通路使相邻细胞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通路中的关键成分和相互作用是已知的，但它们如何共同决定细胞的信号状态（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）仍不清楚。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（顺式）相互抑制，而在细胞之间（反式）有效地相互作用以传递信号。细胞发送或接收信号的能力在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，这三种边缘蛋白可以以不同的配体特异性方式改变受体与配体的相互作用强度。因此，即使成分相对较少，细胞也会表现出非直觉的信号状态。为了了解在自然过程中发生的信号状态，以及它们能够实现哪些类型的信号行为，本提案旨在对Notch信号状态如何由受体、配体和边缘蛋白的表达水平决定进行定量和预测性的理解。为此，我们将构建一组细胞系，这些细胞系允许控制配体和边缘表达水平，并读取由此产生的Notch活性。我们将对这些细胞系进行分析，定量评估单个细胞表面同时可用的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来系统地测量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边缘蛋白表达水平和类型的依赖性，以及多个边缘蛋白之间相互作用的影响。我们将开发一个数学建模框架，使用这些数据从组件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培养来分析单个细胞的发送和接收状态。这些方法将允许