Cellular polarization is essential to morphogenesis, immune response, neuronal development, chemotropism, and motility. Saccharomyces cerevisiae is a well-studied model eukaryote that exhibits numerous polarization phenomena, including signal-induced changes in cell shape and nuclear position. During the haploid phase of their life cycle, yeast cells secrete peptide pheromones that transform vegetatively growing cells of opposite mating type into gametes. Preparatory to cellular and nuclear fusion, pheromone triggers polarized growth toward the mating partner (chemotropism), and migration of the nucleus to the tip of the mating projection. Recent results implicate the pheromone-responsive G-alpha protein, Gpa1, in the control of signal-induced polarized growth and nuclear movement. In cells responding to pheromone, Gpa1 interacts directly with the mating-specific MAPK protein, Fus3, and with Kar3, a microtubule motor protein that is essential for nuclear movement during mating. Disruption of the Gpa1-Fus3 interaction confers defects in chemotropism, microtubule orientation, and nuclear migration. The long-term goal of this project is to understand how G alpha proteins regulate the microtubule cytoskeleton in response to external signals. To this end, genetic, biochemical, mass spectrometric, and imaging approaches will be used to determine how Gpa1 affects the mating-specific functions of Kar3, and whether Fus3 is involved in this regulation. Because Gpa1 and Kar3 provide the first example of a G alpha protein/motor-protein interaction, and because G alpha regulation of nuclear movement is unprecedented, this analysis promises to be informative. This project will also provide significant training opportunity. A graduate student and a postdoctoral fellow will be working on several aspects of the research. In addition, a high school student will be recruited to participate in the project each summer.
Scientific Impact Collecting information about the environment, integrating it and responding in accordance with changing conditions are essential functions required of all cells. In eukaryotes, the most common strategy for detecting and transmitting information across the plasma membrane utilizes surface receptors coupled to heterotrimeric G proteins. Animal cells depend on membrane-bound receptors and their associated G proteins to sense hormones, cytokines, neurotransmitters, odorants and light; unicellular eukaryotes use similar molecular switches to communicate with one another and to coordinate developmental fates. An important question is how cells use surface receptors to determine their positions in chemical gradients. Chemotaxis, or directed cell movement in response to a gradient of chemoattractant or repellent, plays a vital role in development and immunity. The related phenomenon of chemotropism — directed cell growth in response to a chemical gradient — is integral to axon guidance, angiogenesis, pollen tube guidance, and fungal infection. Naturally occurring chemical gradients are very shallow and dynamic. How do cells navigate using such subtle cues? The budding yeast Saccharomyces cerevisiae provides an opportunity to study a chemoattractant-sensing G protein-coupled receptor (GPCR) in a model unicellular eukaryote. S. cerevisiae has proven to be of great utility in elucidating many fundamental cellular processes — e.g., cell cycle control, protein trafficking, cytoskeletal dynamics, and signal transduction. During the haploid phase of their life cycle, yeast cells secrete peptide pheromones that bind to GPCRs on cells of the opposite mating type. Preparatory to cellular and nuclear fusion, pheromone triggers polarized growth toward the mating partner, and migration of the nucleus to the tip of the resulting mating projection. The yeast mating response is chemotropic: mating cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. This ability to sense the direction of the gradient source underlies all chemotropic and chemotaxic phenomena. NSF award #0453964 supported research that led to the publication of two significant works, both of which were selected by the American Society for Cell Biology as highlighted papers in the journal, Molecular Biology of the Cell. In the first of these papers, we reported three important observations: (1) The Gα subunit of the mating-specific G protein concentrates (polarizes) on the membrane of the mating projection in cells responding to pheromone; (2) Gα interacts with a microtubule motor protein that is required for movement of the nucleus into the mating projection, the first such interaction discovered; (3) Gα regulates the cortical positioning and attachment of the microtubules that direct and drive nuclear migration during mating. Together, these findings suggested that in addition to its known role in transmembrane signal transduction, Gα serves as a spatial determinant that guides the cell’s directional response. In the second paper, we provided strong evidence that the localization of the pheromone receptor on the plasma membrane changes from uniform to concentrated at the future mating projection site very early in the mating response, before and independent of directed secretion to the growth site. Moreover, we demonstrated that pheromone-induced internalization of the receptor is necessary to polarize its localization, and that in the absence of this process, cells are severely defective in interpreting pheromone gradients. Finally, we showed that pheromone induces the internalization and polarization of the heterotrimeric G protein along with the receptor. Based on these observations, we proposed a model in which polarization of the pheromone receptor is the primary determinant of directional sensing in yeast, and that the coupled trafficking of the receptor and its G protein serves as a positive feedback loop that rapidly amplifies signaling at the growth site. Taken together, the discoveries made over the course of this project suggest a new paradigm in which heterotrimeric G proteins not only act as global regulators of effector enzymes, but also serve as spatial determinants that recruit and amplify signaling complexes at specialized sites. Overall, this project has thus far generated four publications, fostered three collaborations, and laid the groundwork for a detailed understanding of directional sensing in chemotropic cells. Broader Impact During this project period, the PI spearheaded the development of and is now administering the NSF/Capstone Undergraduate Research Program for honors students majoring in Biological Sciences at UIC (see www.uic.edu/honors/learning/bioscapstone.shtml). Ten NSF-sponsored investigators in the PI’s department are participating as mentors in the program. Students are paired with a mentor for a semester of reading followed by four semesters of research leading to a formal presentation of their work at the annual NSF/Capstone mini-symposium. Three NSF/Capstone students worked in the PI’s lab on this project. In all, the research and training of four high school students, two technicians, seven graduate students, and one postdoctoral fellow was made possible by this grant. α
