Project Summary/Abstract Spatial organization and dynamics are the essential features of living systems. Visualizing compartmentalized signaling dynamics is thus critical to understand biological processes. We focus on rational design of new classes of signaling reporters that achieve large dynamic range, high brightness, fast kinetics and high spatial resolution. These reporters are designed based on 1) new physical principles such as multivalent interaction- driven protein phase separation forming intensely bright GFP droplets upon activation of kinases (the reporter is named SPARK for Separation of Phases-based Activity Reporter of Kinase); 2) FlipGFP, a fluorogenic protease reporter by flipping a beta-strand of GFP; 3) SURF (Split UnaG-based Reversible and Fluorogenic) protein-protein interaction (PPI) reporter by splitting and engineering a new fluorescent protein scaffold UnaG by structure-based design and directed evolution. Our primary goal in designing the new classes of signaling reporters is to enable us in visualizing compartmentalized signaling dynamics in living systems, including PPIs that determine specificity of signal transduction, and effectors such as kinases and proteases that regulate all major signaling pathways. If we understand spatial organization and temporal dynamics of signaling, we will be able to better understand molecular mechanisms of biological processes, laying the foundation for understanding disease mechanisms and identifying drugs for the treatment of various diseases including infectious diseases and cancer. While we have made considerable progress, in the next five years, we will address three major unsolved issues: imaging endogenous PPIs, multicolor reporters of kinases and proteases. We will engineer and demonstrate these multicolor and ultrasensitive reporters for multiplex imaging of signaling network in living systems.