INVESTIGATING THE ROLE OF MAPK SIGNALING DYNAMICS AND CHEMOTROPIC REGULATION IN YEAST

MAPK signaling;yeast;mating pathway;biosensor;chemotropism;Biomedical Engineering

Conlon, Patrick JamesLevchenko, Andre ;

Johns Hopkins University

关键词: MAPK signaling;    yeast;    mating pathway;    biosensor;    chemotropism;    Biomedical Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60544/CONLON-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

All eukaryotes possess MAPK pathways which allow cells to respond to diverse environmental stimuli, including stress, mitogenic, and developmental cues. Deviation from strict MAPK signaling regulation has been associated with the development of many human diseases, including cancer and neurodegenerative diseases. To better understand how cells utilize these pathways to make decisions on their fate, researchers often gain valuable insight from studying related pathways in simpler eukaryotic models, such as yeast, due to their genomes being smaller, better defined, and easier to perturb. In this dissertation, we employ new tools to study MAPK signaling dynamics and chemotropic regulation during the pheromone response in Saccharomyces cerevisiae. In the first aim, we examine the role of membrane binding by the MAPK scaffold protein, Ste5, in mediating the chemotropic behavior of cells exposed to gradients of pheromone. Our findings indicate that modulation of scaffold binding to the membrane does not alter directional sensing, but rather sets a chemotropic sensitivity range by modulating the dose response of downstream pathway activity. In the second aim, we explore how the spatial and temporal patterns of Fus3/Kss1 activity contribute to developmental outcomes in cells, including the decision to bud, mate, and undergo polarized morphogenesis in the presence of pheromone. We first apply the Erk Kinase Activity Reporter (EKAR) to specifically report Fus3 and Kss1 activity in live yeast cells. Using this reporter, we are able to elucidate important features of MAPK activity dynamics and cell-to-cell variability. Specifically, we find activity kinetics to exhibit strong cell-cycle dependence and that intracellular activity gradients develop over time as projections grow. In the third aim, we systematically examine mating at the genome-level with the goal of identifying new or previously unidentified mediators of chemotropism in yeast. First, we devised and executed an imaging-based genome-wide screen of mating efficiency and identified groups of mutants exhibiting mating defects. We then explored the nature of the defect in two groups of protein trafficking mutants: the ESCRT proteins and a membrane-localized component of the exocyst complex, Sec3. Our results indicate previously unidentified roles for these proteins in supporting optimal mating pathway activation and chemotropic behavior.