Project Summary Migrating cells use filopodia to interact with and efficiently move through their complex 3D environments. Filopodia are slender actin-filled projections composed of a core of cross-linked, parallel actin bundles. They are highly dynamic, vary in length and found in a wide variety of cell types such as neurons that use them for gradient sensing and efficient directional migration or cancer cells that employ them for moving out from tumors into neighboring tissue. The first steps of filopodia formation are poorly understood. Three conserved proteins are required for their formation - a MyTH4-FERM myosin) and two regulators ofactin polymerization, VASP and Formin. How the action of these three proteins is coordinated to initiate filopodia formation is unknown. The objective of this proposal is to define the molecular mechanism of filopodia initiation with an emphasis on the role of a MF myosin and its functional relationship to the actin polymerase VASP in this process. Recent work revealed that activation and specific targeting of the MF myosin to the cortex requires the actin polymerization activity of the regulator VASP. The versatile model system Dictyostelium will be used to define the mechanism of this collaborative interaction. It will also be used to investigate how the myosin motor and actin regulator work together to organize the fast-growing ends of actin filaments at the membrane to initiate polymerization. A combination of in vivo, in vitro and in silico approaches will be employed to a) gain new insight into the regulation and mechanism of filopodia initiation and filopodial function in vivo; b) characterize the MF myosin motor and its interaction with the actin network in vitro; and c) build a predictive mathematical model of filopodia initiation. The knowledge generated by this project will reveal how cells use a myosin-based motor to build specific actin-based structures such as filopodia. Understanding how initiation occurs will also reveal how cells control filopodia formation to undergo directed migration or invade into surrounding tissues.