The role of dynamics in defining the limits of normal developmental signaling.

Ras/ERK通路的信号传导控制细胞增殖、迁移和凋亡。 分化这一途径的正常运作对人类发展至关重要， 体内平衡该途径中的零星突变在许多癌症中发挥着核心作用，而 遗传性突变导致一系列先天性综合征，称为RASopathies， 认知发育受损、心脏畸形和癌症风险增加。一 一个尚未解决的问题是，正常Ras/ERK信号传导与病理性Ras/ERK信号传导的区别是什么。 已知ERK活性的动态模式--包括强度、频率和蛋白质水平--与ERK活性相关。 其活动的持续时间-是必要的适当的信号。然而，标准方法 测量ERK活性缺乏解决这些基本细节所需的单细胞精度。 在这个项目中，我们将使用活细胞成像，它允许几乎连续的监测， 成千上万的细胞同时，收集数据的music驱动的ERK信号，这是遥远的 比以前更准确和详细。我们将重点关注发现的突变 在RASopathies中，它们作为单一基因驱动病理效应的作用， 发展比癌症更明确，在癌症中，许多其他基因的突变是一个重要的因素。 并发症我们将使用我们的成像平台首次比较ERK的变化， 在单细胞水平上由致病突变引起的信号传导。使用多个体外 系统复制参与发展的细胞过程中，我们将确定如何这些 这些变化改变细胞增殖、迁移和分化。然后我们将解剖 在基因表达水平上这些表型变化的潜在机制，使用一种新的 一类直接整合到人类细胞基因组中的报告基因。职等 激酶动力学，基因表达和细胞行为，我们将量化突变细胞如何响应 多种Ras通路抑制剂，现在被认为是治疗 种族主义。这项工作将产生若干重要成果。首先，它将揭示 与正常功能兼容的信号行为的定量边界，允许我们 了解Ras通路突变如何导致疾病，以及为什么一些突变更容易导致疾病， 比其他人严重。其次，它将使我们能够理性地选择给予哪些药物 对不同突变的患者进行治疗，以便个性化治疗， 每个人的特定信号模式。最后，它将产生一个数学模型， 激酶活性和下游基因表达程序之间的联系， 更好地理解发育程序和工程师所需的细胞反应， 针对激酶活性的现有药物。

Signaling by the Ras/ERK pathway controls cell proliferation, migration, and differentiation. Proper function of this pathway is essential for human development and homeostasis. Sporadic mutations in the pathway play a central role in many cancers, while hereditary mutations cause a series of congenital syndromes, termed RASopathies, which result in impaired cognitive development, cardiac malformations, and increased risk of cancer. A major unanswered question is what differentiates normal from pathological Ras/ERK signaling. It is known that the dynamic pattern of ERK activity – including the strength, frequency, and duration of its activity – are essential to proper signaling. However, the standard methods for measuring ERK activity lack the single-cell precision needed to resolve these essential details. In this project, we will use live-cell imaging, which allows nearly continuous monitoring of thousands of cells simultaneously, to collect data on mutant-driven ERK signaling that is far more accurate and detailed than was previously available. We will focus on the mutations found in the RASopathies, where their role as a single gene driving pathological effects in development is more clearly defined than in cancer, where mutations in many other genes are a complication. We will use our imaging platform to compare for the first time the changes in ERK signaling resulting from disease-causing mutations at the single cell level. Using multiple in vitro systems to replicate cellular processes involved in development, we will determine how these changes modify cell proliferation, migration, and differentiation. We will then dissect the mechanisms underlying these phenotypic changes at the level of gene expression, using a new class of reporters that are integrated directly into the genomes of human cells. At the levels of kinase kinetics, gene expression, and cell behavior, we will quantify how mutant cells respond to multiple Ras pathway inhibitors, which are now being considered as treatments for the RASopathies. This work will have several important outcomes. First, it will reveal the quantitative boundaries of signal behavior that are compatible with normal function, allowing us to understand how Ras pathway mutations lead to disease, and why some mutations are more severe than others. Secondly, it will allow us to make rational choices about which drugs to give to patients with different mutations, so that treatment can be personalized to best normalize each individual's specific signaling patterns. Finally, it will result in a mathematical model of the link between kinase activity and downstream gene expression programs that will allow us to better understand developmental programs and engineer desired cellular responses using existing drugs that target kinase activity.