Spatiotemporal modeling of signal transduction in yeast

描述(申请人提供)：酿酒酵母的交配反应为研究细胞生物学的许多基本方面提供了理想的实验系统，包括信号转导、转录调控、极性建立和梯度传感。当交配型特定信息素结合并激活相反交配型细胞上的G蛋白偶联受体(GPCR)时，这种反应就开始了。然后，该信号通过丝裂原激活的蛋白激酶(MAPK)级联进行传播。MAPK的一个关键功能是启动成功交配所需的遗传程序。在真核细胞中，MAPK介导对生长因子、细胞因子、激素、细胞黏附、压力和营养物质的反应，这些因素决定了广泛的细胞决策过程。信息素诱导的MAPK介导的转录变化不足以进行有效的交配。细胞还必须将生长导向交配伙伴发出的信息素梯度，这一过程被称为趋化性。趋化性要求细胞建立一个前锋(即极化)，并在定向生长过程中保持这个前锋。在所有真核生物中，极性的建立和维持是迁移和分化所必需的，在癌症等疾病中，极性的建立和维持往往变得不受调控。通过将微流控与新型荧光报告程序相结合，我们开发了一种实验系统，使我们能够跟踪暴露于依赖时间的信息素浓度的单个细胞中基因表达的变化和关键极性因素的定位。将我们的微流控平台与数学建模相结合，将使我们能够在系统水平上了解调控转录、细胞周期停滞和极性建立的信号通路。目的1利用时间周期信息素浓度将计算建模和实验分析相结合，以量化调节转录的多个信号基序的相对贡献。对野生型和突变型背景中干扰调控的报告基因表达的定量测量将用于验证或驳斥该模型的基本假设。该目的还测试在信息素信号丢失后的转录持久性是否是一种记忆形式，以促进第二次信息素相遇中的细胞周期停滞和极化。目的2将这一综合研究策略扩展到剖析调节极性建立的时空动力学的反馈回路。再一次，这些实验将被设计来告知和验证我们的数学模型。我们研究的最终目标是生成活体细胞过程的真正可预测的模型，并为将研究策略扩展到人类细胞中的信号网络提供路线图，从而允许设计治疗疾病的新方法的基本原理。

DESCRIPTION (provided by applicant): The mating response of the yeast Saccharomyces cerevisiae provides an ideal experimental system for studying many fundamental aspects of cell biology, including signal transduction, transcriptional regulation, polarity establishment, and gradient sensing. The response is initiated when a mating-type specific pheromone binds to and activates a G-protein coupled receptor (GPCR) on a cell of opposite mating type. The signal is then propagated by a mitogen activated protein kinase (MAPK) cascade. A key function of the MAPK is to initiate the genetic program required for successful mating. In eukaryotic cells, MAPKs mediate responses to growth factors, cytokines, hormones, cell adhesion, stress and nutrients that determine a wide range of cellular decision processes. The MAPK mediated alterations in transcription induced by pheromone are not sufficient for efficient mating. Cells also must orient growth toward a pheromone gradient emanating from a mating partner, a process referred to as chemotropism. Chemotropism requires that cells establish a front (i.e., polarize) and that this front is maintained during directed growth. Polarity establishment and maintenance are required for migration and differentiation in all eukaryotes, and often become dysregulated in diseases, such as cancer. By combining microfluidics with novel fluorescent reporters, we have developed an experimental system that allows us to track both changes in gene expression and localization of key polarity factors in single cells exposed to time-dependent pheromone concentrations. Integrating our microfluidic platform with mathematical modeling will enable a systems-level understanding of the signaling pathways that regulate transcription, cell cycle arrest, and polarity establishment. Aim 1 combines computational modeling and experimental analyses using temporally periodic pheromone concentrations to quantify the relative contributions of the multiple signaling motifs that regulate transcription. Quantitative measurements of reporter gene expression in wild-type and mutant backgrounds that perturb regulation will be used to validate or refute the underlying hypotheses of the model. The aim also tests whether transcriptional persistence following loss of pheromone signal is a form of memory to advance cell cycle arrest and polarization in a second pheromone encounter. Aim 2 extends this integrated research strategy to dissect the feedback loops that regulate the spatiotemporal dynamics of polarity establishment. Again the experiments will be designed to inform and validate our mathematical models. The ultimate goals of our investigations are to generate truly predictive models of in vivo cellular processes and provide a roadmap for extending research strategies to signaling networks in human cells, allowing the rationale design of new approaches for treating disease.