Abstract
The establishment and maintenance of polarity is a critical process that is required for numerous cellular functions in eukaryotic cells from single celled yeast to complex mammalian systems. One of the primary drivers of cellular polarity is the actin cytoskeleton, a conserved, polarized polymer system that plays a fundamental role in such processes as cell migration, intracellular transport, cell morphogenesis, and cytokinesis. The highly dynamic nature of filamentous actin (f-actin) allows cells to rapidly respond to external and internal cues and re-orient cell polarity and restructure cell shape at will. In order to rapidly remodel actin networks in vivo, cells employ an array of assembly and disassembly factors. This thesis work has focused on the regulation of actin cytoskeleton organization in the budding yeast Saccharomyces cerevisiae, which can divide mitotically as either haploids or diploids. In addition, haploid yeast of opposite mating types secrete pheromones to attract each other and enter the mating response pathway, where they build a mating projection, fuse to form diploids, and later undergo meiosis and sporulation to form halpoids again.As introduced in Chapter 1, throughout all mitotic and reproductive phases of the yeast life cycle, there are two distinct, prominent actin structures: endocytic actin patches and polarized actin cables. The actin cables are continuously extended from the bud tip and neck and are disassembled closer to the back of the mother cells. They serve as linear tracks for myosin-based delivery of secretory vesicles and other cargo to polarity sites, where they undergo exocytosis. These vesicles carry cell wall remodeling enzymes and other molecular ingredients that allow yeast cells to expand and reshape their cell wall, enabling polarized cell growth and division. Actin cables are assembled by two formin proteins, Bni1 and Bnr1, which nucleate actin assembly at polarity sites (bud tip and bud neck, respectively) and remain processively attached to the barbed ends of filaments as they elongate. In vivo, the activities of Bni1 and Bnr1 must be tightly controlled in order to assemble an organized array of actin cables that is able to efficiently facilitate secretory traffic. This regulation is achieved by a number of formin-binding proteins, which positively or negatively regulate different aspects of formin activities on actin filaments. In this thesis, I characterize a new formin regulator, which I named Bud6 Interacting Ligand 2 (Bil2). I show that Bil2 has distinct regulatory effects on Bni1 and Bnr1 in vitro and in vivo.
In Chapter 2, using a combination of genetics, biochemistry, and cell biology, I demonstrate that Bil2 strongly inhibits the actin nucleation activity of Bnr1 (but not Bn1). Loss of BIL2 results in excessive and disorganized actin cables produced by Bnr1, and leads to defects in secretory vesicle traffic. These observations are supported by TIRF microscopy experiments showing that Bil2 strongly inhibits Bnr1-mediated actin nucleation but not elongation. Consistent with these biochemical observations, I show a deletion of BIL2 is synthetic lethal with a deletion of HOF1, the only other known inhibitor of Bnr1-mediated actin nucleation. Finally I show that Bil2 localizes to the bud neck, the cytosol, and secretory vesicles, and discuss the implications of this localization pattern for Bil2 in vivo functions.
In Chapter 3, I show that Bil2 plays a critical role spatially regulating Bni1 during the yeast mating response, where Bni1 appears to be the only active formin. When exposed to mating pheromones, bil2∆ cells aberrantly form additional mating projections, and simultaneously assemble actin cables from both mating projection tips. Further, Bni1-GFP is tightly focused at mating projection tips in wildtype cells, but depolarized and dispersed in bil2∆ cells. In vitro, Bil2, together with its binding partner Bud6, cluster Bni1 molecules into nucleation centers, producing aster-like actin structures. The arrangement of these asters is very similar to how actin cables are organized in vivo. In both the bud compartment of mitotically dividing cells and in the mating projections of cells responding to pheromone, Bni1 is tightly focused in a spot, and the pointed ends of actin cables extend away from this spot. Thus, my in vitro assays using purified proteins successfully reconstitute key aspects of Bni1 spatial regulation in vivo.
Together, the results presented in this thesis provide new insights into how each of yeast formins, Bni1 and Bnr1, are spatially and temporally regulated in vivo to control actin cable assembly and polarized secretion. Further, they identify Bil2 as the first cellular factor to exhibit distinct regulatory effects on two different formins.