SINGLE CELL ASSAYS TO UNDERSTAND SIGNALING NETWORKS

描述：(申请人摘要)本提案有两个总体目标： 第一个目标是开发和应用一种新的研究策略 结合全基因组范围的所有信号蛋白的功能基因组学 用单细胞信号分析来破解基本的 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， 大卫博士指导下的生物信息学和信号转导 波特斯坦和托拜厄斯·迈耶。应聘者目前拥有丰富的经验 在开发生物仪器方面，想用K25 职业奖将成为研究信号转导的终身教职员工 从功能基因组学的角度看网络。 有越来越多的证据表明存在串扰和反馈 信号通路，特别是在那些参与生长和 可能涉及数千种基因产物的分化。信令 路径不再被认为是独立的、线性的事件集，但 相反，必须将其理解为一个动态网络。虽然目前有 通过出色的分析确定网络中不同玩家的身份 -例如，通过酵母双杂交筛选-或通过以下方式获得最终读数 使用微阵列，缺乏研究网络的工具 动态地，并理解玩家如何在 不同的受体刺激。 候选人最近共同开发了一种逝去波单电池阵列 宏观(E-SCAM)，并用它来显示蛋白质的时间过程 移位和激活可以在数千个单细胞中测量到 同时。通过继续开发这个用于监测的E-SCAM系统 随时间推移的多个信令事件，以及量化 扰乱了这样的网络，拟议的工作就能建立起来 信令网络参数与网络参数之间的定量动力学关系 开始建立细胞信号转导网络如何运作的模型。 选择PDGF刺激NIH-3T3成纤维细胞作为模型系统 所产生的信令响应的复杂性是确定的，并且由于 许多互补的实验方法已经提供了数据，这些数据将 对拟议的研究很有用。

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.