Grant: Catalyst Grant: Invention and High-Risk, High-Benefit Research

Very few of the molecular tools employed in biomedical research have had greater impact than intrinsically fluorescent proteins from marine organisms. Indeed, the 2008 Nobel Prize in Chemistry was awarded to the pioneering researchers who discovered and developed these now ubiquitous tools. The availability of these colorful proteins allows biologists to use well-established molecular biology techniques to introduce a fluorescent label into cells or tissues in a chemically precise manner. With the label in place, the localization of specific proteins is revealed by a bright fluorescence glow that can be readily visualized using specialized microscopes. While labeling applications are the primary use of fluorescent proteins, these robust and versatile tools can also be used for the creation of biosensors that can actively report on biochemical activities in the cell. For example, biosensors based on fluorescent proteins can report on the concentration of important ions or molecules or on the activity of particular enzymes. Our research group has recently used protein engineering to develop a set of new fluorescent proteins that enable us to image two biosensors inside of a single cell. This breakthrough has opened the door to a wide variety of new research applications since we can now monitor two biochemical activities simultaneously to investigate causality and kinetics in individual cells. We now propose to construct a series of new biosensors that will allow us to image intracellular calcium ion concentrations in live cells with an unprecedented level of spatiotemporal precision. This work will produce a toolbox of dual biosensors that will be accessible to all researchers. From this toolbox, a researcher will be able to choose a binary combination of biosensors and investigate important biological questions from a new perspective, ultimately leading to medical breakthroughs in our understanding of debilitating diseases such as cancer and diabetes.