SINGLE CELL ASSAYS TO UNDERSTAND SIGNALING NETWORKS

（申请人的摘要）本提案具有两个总体目标： 第一个目标是制定和应用一项新的研究战略， 功能基因组学通过结合所有信号蛋白的全基因组视图 单细胞信号分析，以打破基本的 细胞信号转导网络的机制。第二个目标是 在基因组学领域给予候选人一流的培训， 生物信息学和信号转导博士的指导下.大卫 Botstein和Tobias Meyer。候选人目前具有丰富的经验 在生物仪器的发展，并希望使用K25 职业奖成为终身教职的教师调查信号转导 从功能基因组学的角度来看。 有越来越多的证据表明，存在的串扰和反馈， 信号通路，特别是那些参与生长和 分化，其中可能涉及数千种基因产物。信令 路径不再被认为是独立的、线性的事件集合， 而是必须理解为一个动态网络。虽然目前有 用于确定网络中不同参与者身份的出色分析 - 例如，通过酵母双杂交筛选，或者通过 使用微阵列，缺乏研究网络的工具， 动态地，并了解玩家如何互动的背景下， 不同的受体刺激。 候选人最近共同开发了一种倏逝波单细胞阵列 Macroscope（E-SCAM），并用它来显示蛋白质的时间过程 可以在数千个单细胞中测量易位和活化 同步通过继续开发这个电子诈骗系统， 随着时间的推移，多个信令事件，沿着方法， 扰动这样一个网络，拟议的工作将能够建立 信号网络参数之间的定量动力学关系 开始生成细胞信号转导网络如何运作的模型。 选择NIH-3 T3成纤维细胞的PDGF刺激作为模型系统，因为 所得到的信令响应的复杂性是公认的 许多互补的实验方法提供了数据， 对拟议的研究有用。

DESCRIPTION: (Applicant's Abstract) This proposal has two overall objectives: The first objective is to develop and apply a new research strategy in functional genomics by combining a genome wide view of all signaling proteins with single cell signaling assays in order to break down the fundamental mechanisms of cellular signal transduction networks. The second objective is to give the candidate first-rate training in the fields of Genomics, Bioinformatics, and Signal Transduction under the guidance of Drs. David Botstein and Tobias Meyer. The candidate currently has significant experience in the development of biological instrumentation and would like to use the K25 Career Award to become a tenure track faculty investigating signal transduction networks from a functional genomics perspective. There is more and more evidence for the existence of cross-talk and feedback in signaling pathways, particularly in those involved in growth and differentiation where several thousand gene products may be involved. Signaling pathways can no longer be thought of as independent, linear sets of events, but rather must be understood as a dynamic network. While there are presently excellent assays to establish the identity of different players in the network - for example, by yeast two-hybrid screens - or to obtain final readouts by using microarrays, there is a lack of tools with which to study networks dynamically and to understand how the players interact in the context of different receptor stimuli. The candidate has recently co-developed an Evanescent-wave Single Cell Array Macroscope (E-SCAM) and has used it to show that timecourses of protein translocation and activation can be measured in thousands of single cells simultaneously. By continuing to develop this E-SCAM system for monitoring multiple signaling events over time, along with methodology to quantitatively perturb such a network, the proposed work will be able to establish quantitative kinetic relationships between signaling network parameters and begin to generate models of how cellular signal transduction networks function. PDGF-stimulation of NIH-3T3 fibroblasts was chosen as a model system since the complexity of the resulting signaling responses is well established and since many complementary experimental approaches have provided data that will be useful for the proposed study.