SINGLE CELL ASSAYS TO UNDERSTAND SIGNALING NETWORKS

通过单细胞分析了解信号网络

• 批准号: 6490430
• 负责人: Mary N Teruel
• 金额: $ 14.37万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490430
• 关键词: 3T3 cells; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biological information processing; biological signal transduction; biological transport; biomedical equipment development; charge coupled device camera; chemical kinetics; computer program /software; computer system design /evaluation; data collection methodology /evaluation; electrophysiology; fluorescence microscopy; fluorescent dye /probe; functional /structural genomics; green fluorescent proteins; intermolecular interaction; microarray technology; platelet derived growth factor; receptor coupling; second messengers; single cell analysis; time resolved data

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.

描述：（申请人摘要）该提案有两个总体目标： 第一个目标是制定并应用新的研究策略 功能基因组学，结合所有信号蛋白的基因组范围 通过单细胞信号传导分析来分解基本的 细胞信号转导网络的机制。第二个目标是 为候选人提供基因组学领域的一流培训， 在博士的指导下生物信息学和信号转导。大卫 博特斯坦和托比亚斯·迈耶。候选人目前拥有丰富的经验 正在开发生物仪器并希望使用 K25 职业奖成为研究信号转导的终身教授 从功能基因组学的角度来看网络。 越来越多的证据表明串扰和反馈的存在 信号通路，特别是那些涉及生长和发育的信号通路 可能涉及数千个基因产物的分化。信令 路径不再被认为是独立的、线性的事件集，而是 相反，必须将其理解为动态网络。虽然目前有 出色的分析方法可确定网络中不同参与者的身份 - 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 动态地了解玩家如何在环境中互动 不同的受体刺激。 该候选人最近共同开发了倏逝波单细胞阵列 宏观显微镜 (E-SCAM) 并用它来表明蛋白质的时间进程 可以在数千个单细胞中测量易位和激活 同时地。通过继续开发这个E-SCAM系统进行监控 随着时间的推移发生多个信号事件，以及定量方法 扰动这样一个网络，建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。 NIH-3T3 成纤维细胞的 PDGF 刺激被选为模型系统，因为 由此产生的信号响应的复杂性是众所周知的，因为 许多互补的实验方法提供了数据，这些数据将被 对拟议的研究有用。