Breshears, Laura Marie2009-10-142009-10-142009-08https://hdl.handle.net/11299/54107University of Minnesota Ph.D. dissertation. August 2009. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Dr. Margaret A. Titus. 1 computer file (PDF); x, 132 pages, appendix (pages 92-123)Directed cell migration (chemotaxis) is a fundamental biological process necessary for embryonic development, wound healing, and proper function of the immune system. Chemotaxis also plays a significant role in many developmental disorders and post-embryonic diseases in humans, such as cancer. Chemotaxis is driven by extracellular cues that act, in large part, to induce changes in the actin cytoskeleton, such as actin polymerization, that facilitate directed cell migration. Myosins are actin-associated motors that have a variety of functions in different cellular contexts. Myosins can effect cortical tension, pseudopod and filopodia formation, phagocytosis, the function of sensory structures, and the basic mechanics of cell motility. Members of the MyTH/FERM family of unconventional myosins all have roles in actin-based processes and one member, vertebrate myosin X, has recently been shown to play a role in actin dynamics in response to extracellular migration cues. The social amoeba Dictyostelium discoideum is a powerful model system for dissecting chemoattractant signaling pathways and identifying the cytoskeletal components necessary for directed cell migration. MyoG is a novel unconventional myosin characterized by two MyTH/FERM domains in its tail region. The potential role of this myosin in Dictyostelium cell migration was investigated by analyzing the phenotype of three independent myoG null mutants. The initial stages of Dictyostelium development, induced by starvation, depend on chemotaxis to cAMP, resulting in the formation of a multi-cellular aggregate. Upon starvation myoG — cells fail to aggregate, arresting as a smooth monolayer of cells. The myoG — cells neither polarize in a cAMP gradient nor do they chemotax toward the cAMP source. Analysis of the ability of myoG — cells to polymerize actin in response to cAMP revealed that the response is dampened in the mutants. myoG — cells are also defective in signaling to PI3K in response to cAMP. These data show that while the mutant cells retain some ability to respond to the gradient, the major pathways regulating polarity and chemotaxis are not functional. The mutant phenotype suggests that MyoG acts in transducing the chemotactic signal from the cAMP receptor to PI3K and the actin cytoskeleton, facilitating the morphological changes that lead to polarization and directional migration. The role of MyoG in chemotactic signaling represents a novel function for an unconventional myosin. The work presented here clearly demonstrates that MyoG is necessary for signaling from the cAMP receptor to both PI3K and the actin cytoskeleton. Sequence analysis shows that there is no direct homologue of MyoG in other organisms, but the high degree of conservation of the chemotactic signaling pathways indicates that there are likely to be functional homologues in higher eukaryotic cells, such as neutrophils, that rely on chemotaxis for cellular function.en-USActinChemotaxisCytoskeletonDevelopmentDictyosteliumMyosinMolecular, Cellular, Developmental Biology and GeneticsThesis or Dissertation