Dynamics of Signaling Pathways: Mechanism and Function

描述(申请人提供)：我们的长期目标是了解生物信号的动态行为是如何被控制的，以及这些动态是如何影响细胞反应的。这项建议侧重于P53信号通路的基本细胞机制。我们最近对单细胞进行了长期延时成像研究，发现P53水平对特定类型的DNA损伤表现出高度意想不到的脉动性反应。这些重复的脉冲在之前测量细胞群体中P53水平的研究中被掩盖了。我们将结合单个活细胞的定量动态测量、数学建模和对P53电路的操作来询问P53信号通路如何以及为什么产生这一系列统一的脉冲，以及为什么不同的细胞显示不同数量的脉冲。我们的第一个目标是确定导致P53脉冲的分子机制以及控制其幅度和持续时间的反馈。我们将对P53途径中几种蛋白质的动态进行定量的群体和单细胞测量，并将这些信息整合到一个理论框架中，目标是开发一个可信的P53动态预测模型。接下来，我们将确定P53的脉动行为如何与特定的细胞结果以及特定下游程序的激活(如细胞凋亡、细胞周期停滞和DNA修复)联系在一起。我们将跟踪P53的动态，同时追踪报告单个活细胞下游程序的标记蛋白，并确定每个成像细胞的命运。我们将操纵控制电路来改变脉冲的频率或幅度，或者完全消除它们，并询问这些变化如何影响细胞的结果。除了询问P53的动态如何决定结果，我们还将检查DNA损伤量是否影响脉冲的数量。我们已经开发了一种新的系统来定量活细胞中的DNA双链断裂(DSB)和细胞周期阶段，我们将使用该系统与跟踪P53脉冲并行使用，以研究DSB的初始数量是否影响P53脉冲的数量，以及细胞对辐射的敏感性是否在细胞周期中发生变化。最后，我们将确定在携带MDM2启动子(SNP309)特定多态的细胞中，雌激素如何影响P53的动力学和DNA损伤后的细胞命运，这些细胞被证明具有高水平的MDM2而没有P53脉冲。我们将以SNP309细胞为背景，预测和测试雌激素拮抗剂和P53/MDM2抑制剂对P53动态的影响。我们的结果将为控制和操纵P53途径提供新的见解，P53途径可能是保护人类细胞免受癌症发展的最重要途径。这些研究将对p53的生物学机制和功能有更深入的了解，并将为分析、描述和理解单个活细胞中其他信号通路的动力学提供一个原型。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how the dynamic behavior of biological signals is controlled and how these dynamics affect cellular responses. This proposal focuses on fundamental cellular mechanisms of the p53 signaling pathway. We recently used long-term time-lapse imaging studies on single cells and discovered that p53 levels show a highly unexpected pulsatile response to specific types of DNA damage. These repeated pulses had been masked in previous studies that measured p53 levels in populations of cells. We will combine quantitative dynamic measurements in single living cells, mathematical modeling and manipulation of the p53 circuit to ask how, and why, the p53 signaling pathway generates this series of uniform pulses and why different cells show different numbers of pulses. Our first aim is to identify the molecular mechanism leading to p53 pulses and the feedbacks that control their amplitude and duration. We will perform quantitative population and single cell measurements of the dynamics of several proteins in the p53 pathway and integrate the information into a theoretical framework, with the goal of developing a credible predictive model for p53 dynamics. Next, we will determine how p53 pulsatile behavior is connected with specific cellular outcomes and with the activation of specific downstream programs such as apoptosis, cell cycle arrest and DNA repair. We will track p53 dynamics in parallel with marker proteins that report on downstream programs in single living cells, and identify the fate of each imaged cell. We will manipulate the control circuit to alter the frequency or amplitude of the pulses, or eliminate them altogether, and ask how these changes affect the outcome for the cell. As well as asking how p53 dynamics determine outcome, we will examine whether the amount of DNA damage affects the number of pulses. We have developed a novel system for quantitating DNA double- stranded breaks (DSBs) and cell cycle stage in live cells and we will use this system in parallel with tracking p53 pulses to ask whether the initial number of DSBs affects the number of p53 pulses, and whether the cell's sensitivity to radiation changes during the cell cycle. Finally, we will determine how estrogen influences p53 dynamics and cell fate following DNA damage in cells that carry a specific polymorphism in Mdm2 promoter (SNP309), which were shown to have high levels of Mdm2 and no p53 pulses. We will predict and test the effect of estrogen antagonists and p53/Mdm2 inhibitors on p53 dynamics in the background of SNP309 cells. Our results will provide new insights into the control and manipulation of the p53 pathway, perhaps the most important pathway protecting human cells against the development of cancer. These studies will give a deeper understanding of the biological mechanisms and function of p53, and will provide a prototype for the analysis, description, and understanding of the dynamics of other signaling pathways in single living human cells.