PROJECT SUMMARY The regulation of the actin cytoskeleton facilitates changes in cell shape and directed motility and is essential for processes such as embryogenesis, wound healing, and cancer invasiveness. A thorough understanding of the control of cell shape will require a more comprehensive characterization of how the cellular actin architecture is dynamically regulated in both normal and pathological contexts, which in turn will inform therapeutic strategies that target actin dynamics. The highly conserved Arp2/3 complex plays an essential role in nucleating networks of branched actin filaments that govern an array of cellular behaviors. Using highly tractable cell lines derived from an Arpc2 conditional knockout mouse, we are poised to use innovative experimental approaches alongside established techniques to elucidate novel aspects of Arp2/3 and branched actin regulation. These efforts will provide a more comprehensive view of the control of cellular behavior, both in terms of depth, with focus on spatiotemporal dynamics of actin structure and regulators, as well as breadth, by identifying novel actin regulators and cell signaling feedback mechanisms.