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

描述（由申请人提供）：促分裂原活化蛋白激酶（MARK）通路是调节多种细胞过程的进化保守的真核细胞信号传导模块。响应于多种细胞外刺激，MAPK磷酸化并激活下游转录因子，其修饰染色质并协调基因表达。许多MARK途径已在酿酒酵母中被遗传定义。在这个建议中，我们专注于一个良好的特点酵母MARK途径，高渗透压甘油（HOG）的反应途径。在过去的几十年中，该级联反应的组分和调控网络已经使用在大细胞群体上进行的遗传和生物化学测定来阐明。在这个建议中，我们采取了一种补充的方法，通过监测信号在单细胞中的动态与高时间分辨率。（1）MARK活性将在活细胞中测量;（2）测量几乎是瞬时的，给出1秒量级的时间分辨率，这是单个反应步骤动力学的典型时间尺度;（3）将在许多单个细胞上并行执行测量，允许研究细胞与细胞信号动力学的可变性，并允许直接测量网络中关键组分的浓度与细胞中信号动力学之间的相关性。单细胞具体的目标是围绕测量信号动力学在三个不同的时间尺度：（1）反馈调节激活之前的初始快速信号传播;（2）反馈调节主导的适应动力学和（3）进化的时间尺度，这将通过使用相关酵母物种的HOG信号通路的比较实验分析来探索。拟议的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。与公共卫生的相关性：一些哺乳动物MAPKs被炎性细胞因子和环境应激激活，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，因此提出的实验和理论技术和概念对于高等真核生物中MARK级联的分析将非常重要。

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.