Biological Reaction Networks Characterized by Total Internal Reflection with Fluorescence Correlation Spectroscopy

荧光相关光谱全内反射表征的生物反应网络

• 批准号: 0641087
• 负责人: Nancy Thompson
• 金额: $ 78.71万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0641087&HistoricalAwards=false
• 关键词: Biological; Reaction; Networks; Characterized; Total

Many if not all biological processes in eukaryotic cells involve at some point signaling in response to ligand-receptor interactions. One characteristic of these ubiquitous processes is that they often employ multiple interacting components. Quantitative analysis and modeling of such reaction networks is at present severely restricted by the lack of high-throughput methods for measuring the thermodynamic and kinetic parameters of the individual interactions composing the networks. Thus, there is an urgent need for techniques that can address this very widespread impasse. The goal of this research project is to combine total internal reflection fluorescence microscopy (TIRFM) with fluorescence correlation spectroscopy (FCS) as a method for addressing this limitation. One of the key features of this approach is that it is predicted to be able to provide kinetic (and not just thermodynamic) information about nonfluorescent molecules participating in the network. The PI is in a unique position to pursue the development of methods in which these two techniques are combined, given her previous extensive experience in both TIRFM and FCS. Two test systems will be explored: the mouse Fc receptor (FcRII) with its interaction partners and the human nuclear pregnane X receptor (PXR) with its interaction partners. The PI has considerable prior experience with the first system. For the second system, Prof. Matthew Redinbo of the PI's department, an expert on the structure and function of PXR, will act as a full collaborator. A key aspect of this research is the use of microfluidic devices along with a fast EMCCD. For the design, construction, and implementation of microfludic chambers, Prof. J. Michael Ramsey, a colleague in the PI's department and an expert in the design of such devices, will also collaborate. Two immediate, biologically relevant results of this project will include increased understanding of the manner in which FcRII functions, providing a paradigm for other cell-surface associated immune reactions, and increased understanding of the manner in which PXR functions, providing a paradigm for other nuclear receptors. In both cases, there is at present almost no quantitative information available about how the different interactions involved combine as a network. Perhaps more importantly, because instrumentation for both TIRFM and FCS are now commercially available and are being increasingly acquired by other laboratories, it is expected that the developed methods will find wide application in the biological sciences.The PI is, and has been for many years, actively involved in classroom teaching as well as training numerous postdoctoral associates, graduate students and undergraduate students in her research laboratory. In addition, the PI has spent considerable effort on recruiting women and members of under-represented minorities to science and improving their working conditions. This research project, although it will provide new paradigms for the methods by which immune and nuclear receptors function, is also focused on the development of TIR-FCS as a new, high-throughput method for measuring the thermodynamic and kinetic parameters required for adequate understanding of biochemical networks, a current impasse in biology. The PI will close this gap and, accordingly, all developed data acquisition and analysis software will be made freely available through the internet.

真核细胞中的许多(如果不是全部)生物过程在某个时刻涉及到响应配体-受体相互作用的信号传递。这些无处不在的流程的一个特征是它们经常使用多个交互组件。目前，由于缺乏高通量方法来测量组成网络的各个相互作用的热力学和动力学参数，对这种反应网络的定量分析和建模受到严重限制。因此，迫切需要能够解决这一非常广泛的僵局的技术。本研究项目的目标是将全内反射荧光显微镜(TIRFM)和荧光相关光谱(FCS)相结合，作为解决这一局限性的方法。这种方法的关键特征之一是，它被预测能够提供关于参与网络的非荧光分子的动力学(而不仅仅是热力学)信息。鉴于她之前在TIRFM和FCS方面的丰富经验，PI在开发这两种技术相结合的方法方面具有独特的地位。将探索两个测试系统：小鼠Fc受体(FcRII)及其相互作用伙伴和人核孕烷X受体(PXR)及其相互作用伙伴。PI对第一个系统有相当多的经验。对于第二个系统，PI系的Matthew Redinbo教授是PXR结构和功能方面的专家，他将作为全面的合作者。这项研究的一个关键方面是使用微流控器件和快速EMCCD。对于微流体室的设计、建造和实施，PI部门的同事、此类设备的设计专家J.Michael Ramsey教授也将合作。该项目的两个直接的生物学相关成果将包括增加对FcRII功能方式的理解，为其他细胞表面相关免疫反应提供一个范例，以及增加对PXR功能方式的理解，为其他核受体提供一个范例。在这两种情况下，目前几乎没有关于所涉及的不同相互作用如何组合成网络的定量信息。也许更重要的是，由于TIRFM和FCS的仪器现在已经商业化，并越来越多地被其他实验室获得，预计所开发的方法将在生物科学中得到广泛应用。PI多年来一直积极参与课堂教学，并在她的研究实验室培训无数博士后助理、研究生和本科生。此外，国际和平协会在招募妇女和代表性不足的少数群体成员进入科学界并改善他们的工作条件方面做出了相当大的努力。这项研究项目虽然将为免疫和核受体发挥作用的方法提供新的范式，但也专注于开发TIR-FCS作为一种新的高通量方法，用于测量充分理解生化网络所需的热力学和动力学参数，这是目前生物学中的一个僵局。PI将缩小这一差距，因此，所有开发的数据采集和分析软件都将通过互联网免费提供。