In this project, we will dissect the roles of class I myosins, molecular motors that bind actin filaments through their motor domains and plasma membrane through their tail domains, in actin-mediated cell shape changes. We will test the hypothesis that myosin I motor activity promotes assembly of branched actin networks at the sites of membrane deformation, including endocytic vesicles, phagocytic cups, and podosomes. The proposed work will use two model systems: fission yeast, where myosin I is required for endocytosis, and murine macrophages, which rely on myosin I for phagocytosis and cell migration. In aim 1, we will determine how myosin motor activity contributes to endocytic actin patch assembly and internalization in yeast. We will use the genetically tractable yeast system to express mutant myosin from the endogenous myosin locus and apply molecule counting techniques, which are well established in the yeast endocytosis system, to measure the rates of actin assembly. In aim 2, we will test the role of myosin motor activity in actin and membrane dynamics during phagocytosis and cell migration. These studies will take advantage of the genetically modified mice lacking class I myosins that are available in our labs. In aim 3, we will determine how myosin-membrane interactions are regulated to contribute to myosin functions in actin assembly and immune response. The proposed work will shed light on the molecular mechanisms driving actin- dependent membrane deformation. In order to accomplish these aims, we will replace the spinning disk confocal microscope that is no longer supported by the manufacturer with a state- of-the-art spinning disk microscope suitable for the proposed live-cell imaging studies.