Date of Award
Level of Access Assigned by Author
Doctor of Philosophy (PhD)
Second Committee Member
Third Committee Member
Rebecca Van Beneden
Additional Committee Members
Heterotrimeric G proteins play crucial roles in various signal transduction pathways, where they act as molecular switches in transducing a signal from G protein coupled receptors (GPCRs) at the plasma membrane to downstream effectors. Although their mechanism of action is mostly concentrated at the plasma membrane, their dynamic membrane organization and how it is regulated are not understood. Due to the diffraction limited resolution of fluorescence microscopy, studying the precise organization of membrane proteins can be challenging. In this study, we took advantage of super-resolution fluorescence photoactivation localization microscopy (FPALM) to overcome this challenge. Dictyostelium discoideum was used as a cellular model to study G protein function and membrane organization. These cells rely on chemotaxis toward a secreted chemoattractant, cyclic adenosine monophosphate (cAMP) during the development phase of their life cycle. The Gα2 subunit of D. discoideum is required for the chemotactic response. Once activation occurs, Gα2 is known to be phosphorylated on serine 113; however, the role of this phosphorylation remains poorly defined. Exchange of serine residue 113 to alanine causes starved cells to begin the aggregation phase several hours sooner when compared to wild type, while exchanging this serine to aspartic acid (phosphorylation mimic) shows a dramatic decrease in plasma membrane surface localization. At the nanoscale level, images using FPALM show that activation and phosphorylation cause significant changes to Gα2 cluster density in the plasma membrane. Getting these first nanoscale images of G protein provided robust information, which adds to our understanding of the ligand-dependent reorganization and clustering of Gα2 required for precise signaling. Cell fractionation experiments supported this result. In addition, phosphorylation-dependent interaction between phosphorylated Gα2 and D. discoideum 14-3-3 protein was detected.
Alamer, Sarah A., "Characterization of Phosphorylated G Protein Function and Membrane Culstering by Super Resolution Imaging" (2018). Electronic Theses and Dissertations. 3020.