The manner in which cells monitor their structural organization and coordinate cell morphogenesis and cell division are poorly understood. In yeast two signaling pathways participate in the maintenance of cytoskeletal and cell wall organization: the Hs11p Nim1 related-kinase pathway which is involved in coordinating organization of the septin cytoskeleton with cell cycle progression, and the Slt2p MAP kinase pathway which is important in cellular polarization events during vegetative growth and mating. We plan to elucidate further how these pathways are regulated and function in yeast. Hs11p is a component of a highly conserved cell cycle control pathway, and Hs11p kinase activity depends upon proper organization of the septin cytoskeleton. To understand the mechanism by which Hs11p is regulated during the cell cycle, we will determine how septins and three other proteins that participate in yeast cell morphogenesis, Elm1p, Cla4p, and Hs17p, regulate Hs11p. Additional upstream regulators of Hs11p will be identified, and we will attempt to reconstitute Hs11p activity in vitro. In addition to identifying the mechanisms of Hs11p regulation, we will analyze downstream steps of the Hs11p pathway. We will determine whether Swelp is a direct target of Hs11p, and if Pho85p is a target of the Hs11p-Swe1p pathway. Finally, we will determine if the other members of the Hs11p family, Kinlp and Kin2p, are also involved in the regulation of cell cycle progression. We will also attempt to determine how MAP kinase pathways are linked to the organization of the cytoskeleton. Using the two-hybrid system, we have found that Spa2p interacts with components of two MAP kinase pathways in yeast, the Slt2p pathway and the mating pheromone Fus3p/Kss1p pathway. We will biochemically characterize these interactions, and test if Spa2p serves as a scaffold coordinating these two MAP kinase pathways with cell polarization and actin organization. To further understand how the Slt2p pathway functions, we will also analyze the downstream effectors of this pathway, including Tuslp a novel guanine nucleotide exchange (GEF) factor homologue. Moreover, we have found that the level of the GEF, Rom2p, is regulated by Slt2p; Rom2p activates Rho1p, an upstream regulator of the Slt2p pathway. We will therefore determine if a positive feedback loop regulates the Slt2p pathway. Finally, we will also determine if the Slt2p pathway participates in the septin cytoskeletal checkpoint in yeast. These studies are expected to help elucidate the mechanism by which cells maintain their morphological integrity and spatially and temporally coordinate the organization of the cytoskeleton with nuclear cycle events. Because the components of these pathways are highly conserved, we expect these studies will be of general significance.
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