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

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.