1. Project summary: Actin filaments are essential structural elements in cells and actin filament assembly produces forces for some types of cellular movements such as locomotion and endocytosis. Cellular movements are essential for cell division, embryonic development and defense against microorganisms. Movement of cells out of primary tumors is the chief cause of mortality in cancer. The long-term goal of this research program since its inception in 1972 has been to understand molecular mechanisms that control the dynamics of the actin cytoskeleton. This grant supports basic research on the structures and functions of actin and actin-binding proteins and their roles in live cells. The work focuses on two of two major proteins that regulate actin polymerization, formins and Arp2/3 complex. The formin family of proteins nucleates filaments and cooperates with profilin to regulate their elongation by remaining processively attached to the growing barbed end. These unbranched filaments are used in the lamellar region of migrating cells, actin filament cables in fungi, cytokinetic contractile rings, filopodia and stress fibers. Arp2/3 complex coordinates actin filament assembly during endocytosis and cellular motility with the aid of WASp/Scar proteins (activators of Arp2/3 complex), capping protein (terminator of actin filament elongation), ADF/cofilin (recycles actin subunits) and profilin (nucleotide exchange factor for actin). The fission yeast, S. pombe, is our experimental organism. Motility of its cortical actin patches depends on the same proteins used by motile amoebas and animal cells to advance their leading edges. Fission yeast cells offer outstanding genetics, molecular genetics and spectacular cytology, all of which are required to reach the goals of the project. We manipulate proteins of interest by site directed mutagenesis and study them in live cells after tagging with a fluorescent fusion protein (and verifying their functionality with genetic tests). Methods are available to isolate sufficient quantities of most proteins of interest for structural and biophysical studies. The project has three broad goals for the next four years. The first is to understand how formin FH2 domains influence the structure of the barbed end of an actin filament and how the FH2 domains step reliably onto each new actin subunit added to the filament. The second is to understand how Arp2/3 complex changes its shape from the inactive form free in solution to the active conformation that anchors an actin filament branch to the side of another actin filament. The third is to use high-speed super resolution fluorescence microscopy of live yeast cells and computational modeling to test our hypotheses for the assembly and turnover of the actin filament network at sites of clathrin-mediated endocytosis. We propose to use what we learn from experiments on yeast to image the assembly of individual actin filaments at the leading edge of motile cells.