Chemotaxis occurs during a number of key physiological events including angiogenesis, embryonic development and wound healing. It also contributes to disease progression in pathological conditions such as cancer metastasis and arthritis. The goal of the current proposal is to reveal how biochemical reactions and physical characteristics, such as membrane curvature, deformation, and assembly phase, interact with one another in achieving dynamic, accurate yet highly efficient cell migration. Chemotaxis has been understood mainly in the perspective of signal transduction, while if and how physical properties of membranes play a role, and how they interact with signal transduction remain largely unknown. By newly developing and implementing a series of molecular actuators that can directly probe membrane properties with high spatio-temporal precision inside lively migrating cells, we will reveal an interplay between signal transduction and membrane mechanics. What molecular mechanisms generate local membrane curvatures developing into filopodia and lamellipodia? In sensing chemoattractants, cells polarize by undergoing asymmetric membrane deformation consisting of filopodia and lamellipodia at the front, and membrane retraction at the rear. We recently found that curvature-sensitive proteins are a missing link between actin cytoskeleton and membranes. The result made us hypothesize that actin machinery and curvature sensing and remodeling proteins, when properly modulated in a feedback loop, are sufficient to produce desired types of membrane deformations such as lamellipodia and filopodia. We will thus identify a particular combination of Rho GTPases, actin regulators, and BAR proteins, and the molecular logic thereof, that are responsible for formation of filopodia and lamellipodia. How do signaling components in migrating cells respond to membrane deformation? Migrating cells exhibit dynamic morphological changes at plasma membranes and nuclear envelopes “as a consequence” of cytoskeletal rearrangement regulated by signal components. To explore a possibility that membrane deformation talks back to cytoskeletal and signal components, we will deploy molecular actuators that can directly deform membranes. We will then quantify subsequently emerging activity of signaling components such as receptor tyrosine kinases, PI3K, and small GTPases, as well as transcription factors such as YAP and Elk. How does the phase-separated cytoskeletal biomolecular condensate play a role in membrane deformation? Actin networks can undergo formation of biomolecular condensates at the plasma membrane due to weak multivalent interactions among actin regulators. To examine the physiological importance of such phase separation events, we will adapt molecular techniques to assemble or disassemble the condensates. These operations will uniquely achieve gain- or loss-of function manipulations without altering an amount of the molecular constituents; what is altered is their ph...