Abstract An ongoing revolution in microscopy has revealed previously inaccessible aspects of signaling. These include organization of signaling proteins on dynamic cytoskeletal elements, subtle but important variations in activation kinetics, and information encoded in the oscillation frequencies of signaling circuits. We will explore these regulatory mechanisms by developing novel approaches to visualize and control protein activity, and by using them together in the same cell. Novel image analysis methods and computational modeling applied to multiplexed imaging data will reveal causal connections between signaling events, including those dependent on nested feedback loops. We will pursue new methods to produce biosensors bright enough to study the conformational changes of individual molecules in living cells. On podosomes and adhesion complexes, where important dynamic structures are organized on a scale below the diffraction limit, we will use these new methods to examine the opening of stretch-activated binding sites in real time. Proteins will be controlled with light or small molecules using engineered domains inserted away from the active site, where they can allosterically control activity without perturbing normal interactions. We will study three model systems that are well suited to extract basic principles re the spatio-temporal regulation of signaling: 1) In 'frustrated phagocytosis' macrophages attempt to engulf micropatterned circles. The phagocytic apparatus they build around the circles has precise, reproducible geometry, which will enable us to use quantitative image analysis and simultaneous protein control/visualization to develop mathematical models. 2) We will study how cancer cells move on aligned collagen "superhighways" to find blood vessels. There we will ask how cells use localized signaling to sense and respond to anisotropic mechanical stresses. 3) Finally, cell protrusion/retraction will be used to examine the integration of chemical gradients, mechanical force, and cytoskeletal dynamics. We hope that the behavior of these model systems will reveal ubiquitous signaling mechanisms, and that the new tools can help others explore diverse questions in cell biology.