A Mechanistic Insight into Yeast Chemotropism and the Development of New Tools to Study This Phenomenon
DeFlorio, Reagan E.
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A Mechanistic Insight into Yeast Chemotropism and the Development of New Tools to Study This Phenomenon Reagan E. DeFlorio, Ph.D. Department of Biological Sciences University of Illinois at Chicago Chicago, Illinois (2012) Dissertation Chairperson: Dr. Aixa Alfonso Chemotaxis and chemotropism are important biological processes required throughout eukaryotic development. Understanding how cells interpret complex signaling gradients and direct their movement or growth in response to such gradients is a fundamental question of cell biology. The mating response of Saccharomyces cerevisiae is chemotropic. Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated heterotrimeric G protein. Upon activation by the receptor, Gα dissociates from Gβγ and Gβ is subsequently phosphorylated. Free Gβγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local amplification via the Gβγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. My results show that a mutant form of Gβ that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. My data suggest that phosphorylation of Gβ plays important roles in localized signal amplification and in dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance. Pheromone gradients in mating mixtures are dynamic. Therefore, cells must employ mechanisms to reorient their growth as the gradient direction shifts, but little is know about this process. By following the movement fluorescently-tagged proteins in reorienting cells, I identified Gβ and Spa2 as early regulators of this process, which mark the new site of polarized growth prior to morphogenesis. To advance future studies aimed at elucidating the mechanisms underlying cell reorientation, I developed and validated a novel genetic screen to uncover mutants specifically defective in this process. In collaboration with Dr. Eddington's laboratory at the University of Illinois, I developed a microfluidic device that can generate stable pheromone gradients and rapidly rotate them in 90° increments, mimicking the dynamic gradients in yeast mating mixtures. Collectively, my work has advanced our understanding of the mechanisms guiding yeast chemotropism and generated powerful new tools to facilitate future research of the reorientation process.
G protein Signaling