MAPK Signaling in Single Yeast Cells: Dynamics, Variability, and Evolution

描述（由申请人提供）：丝裂原活化蛋白激酶（MARK）途径是进化保守的真核生物信号传导模块，调节多种细胞过程。在对多种细胞外刺激的响应中，MAPKs磷酸化并激活下游转录因子，这些转录因子修饰染色质并协调基因表达。在酿酒酵母中，许多MARK通路已被遗传学定义。在这篇文章中，我们重点研究了一种表征良好的酵母MARK途径，即高渗透压甘油（HOG）反应途径。在过去的几十年里，这个级联的组成部分和调控网络已经通过对大量细胞进行的遗传和生化分析得到阐明。在这个建议中，我们采取了一种补充的方法，通过监测单细胞的高时间分辨率的信号动力学。与传统的细胞群生化和遗传分析相比，该方法具有三个明显的优势：(1)将在活细胞中测量MARK活性；(2)测量几乎是瞬时的，时间分辨率约为1秒，这是单个反应步骤动力学的典型时间尺度；(3)测量将在许多单个细胞上并行进行，从而可以研究信号动力学中的细胞间变异性，并可以直接测量网络中关键成分的浓度与单个细胞中的信号动力学之间的相关性。具体目标是围绕在三个不同的时间尺度上测量信号动力学来组织的：(1)在反馈调节被激活之前的初始快速信号传播；(2)以反馈调控为主导的适应动力学；(3)进化时间尺度，将通过对相关酵母种HOG信号通路的对比实验分析来探索。该实验工作将与定量建模方法紧密结合，最终目标是建立HOG反应途径的预测模型。与公共卫生相关：一些哺乳动物的MAPKs被炎症细胞因子和环境应激激活，这可能在哮喘和自身免疫等疾病中发挥重要作用。由于MARK级联是高度保守的，所提出的实验和理论技术和概念将对高等真核生物中MARK级联的分析具有重要意义。

DESCRIPTION (provided by applicant): Mitogen-activated protein kinase (MARK) pathways are evolutionary-conserved eukaryotic signaling modules that regulate diverse cellular processes. In response to a multitude of extracellular stimuli, MAPKs phosphorylate and activate downstream transcription factors that modify chromatin and orchestrate gene expression. Many MARK pathways have been genetically defined in Saccharomyces cerevisiae. In this proposal we focus on one of the well-characterized yeast MARK pathways, the high osmolarity glycerol (HOG) response pathway. During the last few decades, the components and the regulatory network of this cascade have been elucidated using genetic and biochemical assays performed on large populations of cells. In this proposal we take a complementary approach by monitoring the signaling dynamics in single cells with high temporal resolution. This method provides three distinct advantages compared to traditional biochemical and genetic assays on cell populations: (1) MARK activity will be measured in live cells; (2) measurements are almost instantaneous giving a temporal resolution on the order of 1 second which is the typical time-scale of the kinetics of individual reaction steps; (3) measurements will be performed on many single cells in parallel, allowing studies of cell-to-cell variability in signaling dynamics and allowing a direct measurement of the correlation between the concentration of a key component in the network and the signaling dynamics in a single cell. The specific aims are organized around measuring the signaling dynamics at three different time-scales: (1) the initial rapid signal propagation before feedback regulation is activated; (2) adaptation dynamics dominated by feedback regulation and (3) evolutionary time-scales, which will be explored by using a comparative experimental analysis of the HOG signaling pathway of related yeast species. The proposed experimental work will be closely integrated with quantitative modeling approaches with the ultimate goal to build a predictive model of the HOG response pathway. Relevance to public health: Some mammalian MAPKs are activated by inflammatory cytokines and environmental stresses which might play an important role in diseases like asthma and autoimmunity. Because MARK cascades are highly conserved, the proposed experimental and theoretical techniques and concepts that will be important for the analysis of MARK cascades in higher eukaryotes.