This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): We propose to continue a nationally visible and responsive center focused on the development of novel fluorescent biosensor and detection technologies for investigating pathways and networks in real time and high spatial resolution in living cells. The renewed center retains the combined experience and infrastructure of two existing centers in Pittsburgh: the Molecular Biosensors and Imaging Center at Carnegie Mellon University and the Center for Biologic Imaging at the University of Pittsburgh. The Technology Development (Core 1) component of the center will create a powerful toolbox of intracellular fluorescent biosensors and reporters that can be used to study many, if not all, of the pathway proteins and activities in living cells. This fluorescent biosensor development program integrates efforts across multiple disciplines, including dye chemistry, molecular biology, biochemistry, structural biology, modeling, cell biology, image acquisition and analysis, and high throughput screening. We have made substantial progress in this effort during the previous 3 years of funding. Four exciting Driving Biology Projects (Core 2) are essential to the technology development effort in Core 1. These DBPs are focused on important and currently un-addressable biological problems, and will serve both as test-beds for the technology and compelling demonstrations of the value of the biosensors and reporters developed in this program. The Infrastructure (Core 3) is provided through the Center for Biologic Imaging at the University of Pittsburgh. The role of the CBI is to act as the application and outreach ami of the project as a whole by testing the new biosensors with challenging biological problems. During the last cycle of this proposal the clear mission of the CBI evolved to become the catalyst in probe implementation, and to strengthen and broaden the impact of the new probes developed by MBIC. This is acheived by providing facilities and expertise to test and validate the probes in the context of the driving biological projects, and ultimately, the biomedical research community at large. The program contains a significant technology transfer component to disseminate concepts, knowledge, software, materials, and resources to users in both academic research labs and industry. This is achieved in our Infrastructure, Training, and Dissemination cores (Core 3,4, 5) and through the technology transfer activities in the Management core (Core 6). RELEVANCE (See instructions): Understanding the regulation of proteins in networks and pathways is central to diagnosing, treating and curing a variety of human diseases. This program develops a new set of tools for observing this regulation process in live cells, as it occurs, and will lead to fundamental advances in technology, biological knowledge, and drug discovery.