Project Summary Many cells can travel from one place to another. This nomadic cell behavior is harnessed to form organs, seal wounds, and hunt down pathogens, but it is often dysregulated in human diseases, including cancer. Consequently, understanding the molecular basis of cell movement has widespread significance for human health. Decades of cell migration studies have outlined how a cell moves on a flat surface. In a two-dimensional environment, a moving cell extends a branched actin-filled protrusion in the direction of movement, while actomyosin contractility retracts the rear. However, cells can move without actin-filled protrusions in vivo using a poorly understood, three-dimensional migration mode called amoeboid migration. Amoeboid migrating cells can move by extruding pressure-filled blebs devoid of actin or rapidly migrate with a near-constant spherical shape using cortical actin flow. Cortical flow-driven amoeboid migration is commonly referred to as fast amoeboid migration (FAM) and is induced by mechanical confinement in a wide variety of cells, with an increased propensity in invasive cancer cells. The non-genetic and extensive induction of FAM, coupled with its use by cancer cells in vivo, necessitates a mechanistic understanding of its underpinnings. However, observing and perturbing FAM is technically challenging in vivo, and the plasticity with which cells can switch how they migrate can confound subsequent analysis. My lab uses the developmental migration of Drosophila primordial germ cells (PGCs) as a facile, reproducible system to study FAM. PGCs natively use FAM in vivo and, importantly, maintain the characteristic cortical actin flows that power FAM outside of the embryo in culture without mechanochemical inputs. Thus, PGCs do not alter how they move, even in a foreign two-dimensional environment. Over the next five years, we will capitalize on the inflexible nature of migratory PGCs to deconstruct FAM and address several outstanding, currently intractable questions: (1) What cortical actin architectures are permissive for cortical flows, and how are these networks assembled, regulated, and maintained? (2) How are cortical flows oriented by guidance cues to direct FAM? (3) How are cortical flows transmitted to a given substrate to generate traction for productive motility? Results from this study will shed light on a poorly understood cell migration strategy and will identify key factors to not only halt it for therapeutic intervention but also endow other cells with its use for cell- based therapies.