PROJECT SUMMARY/ABSTRACT In Eukarya, actin-based protrusions such as filopodia, stereocilia, and microvilli, support a wide variety of cellular functions including motility, mechanosensation, and nutrient absorption. Formation of a surface protrusion involves deformation of the plasma membrane, and previous studies indicate that actin polymerization produces the mechanical force to displace the membrane forward. In addition to the force generated by actin polymerization, protrusion formation may also be powered by other factors. Indeed, the actin-based force generators, myosins, are some of the most abundant residents of actin-based protrusions. Previous research has shown that these motors function as cargo carriers to move critical components needed for assembly to the distal tips. However, only some of the myosins in protrusions are known to carry cargo, while all interact either directly or indirectly with the plasma membrane. As all myosin motors found in actin-based protrusions are also able to exert force, we hypothesize that myosins likely hold the potential to apply a significant impact on the mechanical properties of the membrane. Indeed, based on our preliminary data we have discovered a secondary, cargo-independent pathway for promoting protrusion growth by docking the force generating myosin- 10 motor on the plasma membrane. We hypothesize that myosins exert tipward forces on the plasma membrane to promote actin assembly and protrusion elongation. To test this hypothesize, we will determine how myosin motors drive protrusion elongation by manipulating the mechanical properties of myosin-10, as well as docking additional classes of motor domains on the plasma membrane. Next we will define which modes of membrane attachment impact protrusions elongation by docking the myosin-10 motor domain to three different membrane attachment constructs (i.e. a transmembrane domain, a phosphatidylinositol, or to the negative charge of the inner leaflet of the membrane). Lastly, we will determine how membrane-interacting myosins impact the growing, barbed-ends of filamentous actin (F-actin) assembly by using transmission electron microscopy to visualize the cytoplasmic compartment of the distal tips of protrusions formed in our system. If successful, this research will expand the dogma that myosins assist in protrusion elongation by acting as cargo carriers. This project seeks to understand how membrane-bound myosins drive actin-based protrusions formation in diverse biological settings, which has broad implications for understanding diseases such as Crohn’s disease, deafness, and cancer.