Abstract 1291 Bem One Interacting protein 2 (Boi2) regulates septin organization in the yeast mating projection

G- protein coupled receptors (GPCRs) constitute the largest family of cell surface receptors, orchestrating major physiological processes through mechanisms conserved from yeast to humans in response to extracellular or intracellular signals. In Saccharomyces cerevisiae, yeast GPCRs activate during mating in the presence of mating pheromones, driving polarized, chemotropic growth towards the partner. High pheromone signals induce the formation of a mating projection called a 'shmoo', while low signals prompt elongation and growth towards the source. The intricate process of mating projection formation and gradient tracking relies on septins, which are conserved, cytoskeletal scaffolds involved in cytokinesis and cell polarity. In humans, septin misregulation is associated with neoplasia and neuronal disorders, while in yeast, they lead to defects during mitosis and meiosis. Despite their importance, the molecular mechanism by which septins are spatially controlled remains a mystery. In budding yeast, septin organization is controlled by the active central polarity machinery, Cdc42. Among the Cdc42 associated machinery is the Bem One Interacting protein 2 (Boi2). Boi2 functions in exocytosis, actin regulation and cytokinesis during vegetative growth, with no recognized roles in the pheromone pathway. We hypothesize that Boi2 plays a regulatory role in septin dynamics during the pheromone response pathway. To examine this, we utilized live-cell fluorescence imaging of yeast cells in a microfluidic device, allowing us to track their growth and behavior in real time. Upon examining cells lacking Boi2, we observed aberrant cellular morphology characterized by broad projections and random kinks, indicative of septin defects. Furthermore, our investigations into Boi2 localization during the pheromone response revealed a dose-dependent change in localization from the center of the polarity site (at low doses) to the periphery at the base of the shmoo (at high doses), colocalizing with the septin structures. Subsequently, we utilized a polar cap marker, Bem1GFP along with septin Cdc3-mRuby to scrutinize septin organization in boi2Δ strain. We found that the loss of Boi2 resulted in septin localization to the polar cap rather than the peripheral localization at the base of the shmoo. Boi2 is also known to undergo phosphorylation in response to pheromone at Serine 723, so we examined Boi2 phospho-mutants. Septin structures in S723A mutant exhibited proximity to the polar cap with shorter and smaller shmoos. Conversely, septins in phospho-mimetic S723D mutant were distributed around the cell, farther from the polar cap, coinciding with the formation of longer shmoos. These findings collectively suggest that Boi2 plays a role in the receptor mediated control of septin structure during the pheromone response pathway. This project is funded by NIH-R15, GM140409-01 awarded to Dr. Joshua B Kelley.