Project Summary This proposal focuses on the regulation of formins, a conserved family of actin assembly-promoting proteins that nucleate and processivly elongate actin filaments at their fast-growing barbed ends. Specifically, it uses actin cable formation in S. cerevisiae as a model. While formin structure and function have been defined in some detail, comparatively little is known about how formin activities are spatially and temporally controlled in cells to produce actin structures of a particular shape and size. Recent studies have shown that mammalian homologs of capping protein (CP) and formin can bind simultaneously to the barbed ends of actin filaments, forming ‘decision complexes’ and catalyzing each other’s dissociation to tune actin growth and length1,2. These exciting observations have raised new questions, including whether the decision complex mechanism is conserved in other systems, and whether additional binding partners of formins and/or CP can influence the mechanism. My preliminary data show that yeast CP (Cap1/2) and the yeast formin-binding protein Bud14 work in concert to displace the formin Bnr1 from barbed ends. Thus, my data suggest a novel three-component (Bud14-Bnr1- Cap1/2) decision complex used to regulate the growth of cellular actin structures. In this proposal, I will use in vitro single-molecule imaging to visualize this novel mechanism in real time. Further, I will determine which domains and molecular interactions of Bud14 contribute to the mechanism. In addition, I will use yeast genetics and live-cell imaging to test the three-component mechanism in vivo, and determine how it regulates the proper formation of actin cables with a characteristic shape and length optimized for secretory vesicle traffic. Finally, I will use live-imaging to define the dynamic movements of Bud14 particles in cells, and Bimolecular Fluorescence Complementation (BiFC) assays to study live interactions of Bud14 and Cap1/2 with Bnr1 at the bud neck. Together, this work will provide new insights into how formins are controlled in vivo. This work will expand our understanding of fundamental, conserved mechanisms controlling actin assembly in cells, and provide new insights into the underlying basis of human disease states associated with defects in actin regulation. The specific aims are: (1) Use single-molecule imaging to define the mechanism by which Bud14 and Cap1/2 collaborate to control formin activity at the barbed ends of actin filaments; (2) Test the in vivo role of the Bud14-Bnr1-Cap1/2 mechanism in regulating actin cable length and polarized secretion.