Formins are multidomain proteins that participate in a wide range of cytoskeletal processes that are required for cell polarity, cell migration, cytokinesis, and morphogenesis in all eukaryotes. The defining feature of formin proteins is the Formin Homology 2 (FH2) domain, which directly nucleates actin filaments and remains processively associated with the barbed end of the filament as it grows. The actin assembly activity of formins must be tightly regulated. In the "Diaphanous-related" formins (DRFs), binding of Rho-family GTPases is one mechanism that effects release of autoinhibitory interactions to activate the FH2 domain. Because they reorganize the actin cytoskeleton in response to diverse cellular signals, formins are of central importance in cell biology and to human health. Defects in formin proteins result in failed cytokinesis and abnormal development. Our long-term goal is to understand at a structural level the regulated assembly of actin filaments by formin proteins. We seek to understand the intra- and inter- molecular interactions that regulate formin function using X-ray crystallography and other biophysical and biochemical methods. In the previous project period, we determined crystal structures representing most of the known functional domains of diaphanous-related formins, including the N- terminal regulatory region, and the FH2 and inhibitory DAD domains. In this renewal, we will integrate the structural information provided by these domain fragments to understand the structure of an intact diaphanous related formin, with an emphasis on understanding the autoinhibited state. From this foundation, we expand in two directions to study formins in the active state: (1) dissection of the structural requirements for the nucleation and processive capping activities of the FH2 domain and C- terminal DAD regions, and (2) Structural studies of Bud6 and its interactions with Bni1 and actin. Bud6 is a polarity factor and key binding partner and regulator of formin Bni1 in yeast. Bud6 helps capture microtubule ends at the cell cortex to orient the mitotic spindle. Despite its role at the nexus of interaction between the actin and microtubule-based filaments, its structure remains completely unknown. Collectively, these studies will elucidate regulatory and mechanistic principles common to most or all formins. Our studies of Bud6 will yield a structure-based model of its function in the establishment of cell polarity, and will provide a paradigm for understanding how diverse formins are engaged for specific cellular functions.