| All animal cells require an actin cytoskeleton to maintain their shape and for movement, growth, and replication. The actin cytoskeleton is composed of actin subunits that are assembled into polymers, which come in two distinct forms: three-dimensional networks and unbranched arrays. Three-dimensional networks are nucleated by the Arp2/3-complex and its activators. Chapter 2 involved a study that identified the yeast myosin-I proteins as the second cellular activator of the Arp2/3-complex. This study also showed that a major actin polymerizing machine composed of myosin-I, Vrp1, Bee1/Las17, and the Arp2/3-complex is required for the assembly of yeast actin cortical patches thought to be composed of branched networks.; Chapter 3 and 4 involved studies that established yeast Formins as a major unbranched actin filament nucleator. Formins have been implicated in the regulation of cytoskeletal structures in animals and fungi. Collectively, both chapters showed that the budding yeast Formins, Bni1 and Bnr1, stimulate the assembly of actin filaments in an Arp2/3-independent manner. Furthermore, in vitro, the conserved core of Formin homology domains 1 and 2 of Bni1 (Bni1FH1FH2) was found to nucleate unbranched actin filaments. Bni1FH2 provided the minimal region sufficient for nucleation. Unique among actin nucleators, Bni1FH1FH2 remained associated with the growing barbed ends of filaments. This combination of properties suggests a direct role for Formins in regulating nucleation and polarization of unbranched filamentous actin. Thus, Formins control a distinct pathway to assemble actin filaments that serve to organize the overall polarity of the cell.; The fifth and sixth chapters are mini-chapters that describe two genomic projects I participated in. The first project was based on identifying new actin cortical patch components using protein-protein interaction data predicted by phage-display and determined by two-hybrid analysis for 24 SH3 domain baits. The second project was based on large-scale application of Synthetic Genetic Array (SGA) analysis, a high throughput approach for defining genetic interactions. |
