Project Summary Cellular communication involves the interplay of small molecule signaling species as well as a myriad of enzymatic activities that are tightly regulated in both space and time to orchestrate biological responses. The malfunction of these pathways results in abnormal cellular function and human disease. Our long-term goal is to develop a detailed, molecular level understanding of cellular signaling in both normal and diseased states. We approach this problem through the development and application of designer fluorophore- and protein- based systems. Our lab has developed new classes of fluorescent probes for monitoring and responding to the presence of signaling species associated with human disease. In complementary work, we have developed protein-based approaches for precisely controlling cellular signaling events. During the next five years, we will apply our technologies to illuminate spatial aspects of cell signaling as well as the fine details of the molecular assemblies involved in cell signaling. We will also pursue new platforms for the gated release of agents in living systems and approaches for monitoring signaling enzyme activity in living cells. Together, this work will provide a more detailed understanding of cellular processes associated with human disease.