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：（申请人的摘要）该提案有两个总体目标： 第一个目标是制定和应用新的研究策略 通过结合所有信号蛋白的基因组广泛视图，功能性基因组学 使用单细胞信号测定，以分解基本 细胞信号转导网络的机制。第二个目标是 在基因组学领域进行候选一流的培训， 生物信息学和在DR的指导下的信号转导。大卫 Botstein和Tobias Meyer。候选人目前有丰富的经验 在生物仪器的开发中，想使用K25 成为职业奖，成为统治轨道教师调查信号转导 从功能基因组学角度来看网络。 有越来越多的证据证明存在串扰和反馈 信号通路，特别是在参与增长和 分化可能涉及数千个基因产物。信号 途径不再被认为是独立的线性事件集，但是 而是必须理解为动态网络。虽然现在有 出色的测定方法以确立网络中不同参与者的身份 - 例如，通过酵母双杂交屏幕 - 或通过 使用微阵列，缺乏研究网络的工具 动态并了解玩家如何在 不同的受体刺激。 该候选人最近共同开发了一个evanescent-Wave单细胞阵列 宏观镜（E-SCAM）并用它来表明蛋白质的时间代表 易位和激活可以在数千个单元中测量 同时地。通过继续开发该电子扫描系统以进行监视 随着时间的推移，多个信号事件以及定量的方法 扰动这样的网络，拟议的工作将能够建立 信号网络参数与 开始生成细胞信号转导网络如何功能的模型。 选择NIH-3T3成纤维细胞的PDGF刺激作为模型系统，因为 由此产生的信号响应的复杂性已很好，因为 许多互补的实验方法提供了数据 对拟议的研究有用。