Progress Towards Understanding Neurotransmission: Temporal Mapping of Phospholipase D Activity in Exocytosis

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is supporting the research of Dr. Michelle Knowles at the University of Denver to study a crucial aspect of the transmission of impulses from cell to cell, as in the nervous system. Chemical signals are packed into vesicles in a "sending" cell that are expelled in the direction of a "receiving" cell. Many molecules must organize in space and time to trigger the fusion of a vesicle with the receiving cell membrane. In doing so, cells are capable of releasing hormones and neurotransmitters on demand. The research examines two of these critical fusion-inducing molecules and their relation to one another. This work allows undergraduate and graduate students to be trained in the field of biophysical chemistry where they master cutting-edge technology for observing the behavior of cells. Throughout this project, research and education are integrated into outreach events for students in Denver Public Schools and in the education of non-science majors at the University of Denver. The scientific results of this work are disseminated at scientific conferences and through publications to ensure incorporation of the new found knowledge into the bigger picture of how cells function in the brain and nervous system. The process of membrane fusion requires an input of energy to merge the vesicle lipid bilayer with that of the plasma membrane. In doing so, the membrane quickly transforms from a relatively flat bilayer to a negatively curved tube to form the fusion pore. Although SNARE proteins drive fusion, it is hypothesized that certain lipids aid in the formation of the negatively curved pore. The formation of phosphatidic acid (PA), a lipid that forms negatively curved membranes in vitro, is thought to occur at the fusion site to stabilize the membrane shape changes. Evidence shows that inhibition of the enzyme that makes PA, Phospholipase D1, reduces the amount of PA at the plasma membrane and membrane fusion is blocked. Despite knowing that the presence of PA dramatically affects exocytosis, a clear picture of the timing of PA formation and accumulation at the fusion site is not known. Using high resolution, fluorescence imaging methods, the location of PA and Phospholipase D1 are mapped in time during secretion. This research uses a combination of biochemical reconstitution studies in conjunction with mammalian cell culture to characterize how PA is recruited to the fusion site and how PA affects membrane curvature. Ultimately, the findings determine how PA facilitates exocytosis and lead to a molecular understanding of lipid sorting in cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.