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

有了这个奖项，化学系的生命过程化学项目正在支持丹佛大学的Michelle Knowles博士的研究，以研究神经系统中细胞间脉冲传输的一个关键方面。 化学信号被包装成“发送”细胞中的囊泡，并向“接收”细胞的方向排出。 许多分子必须在空间和时间上组织起来，以触发囊泡与接收细胞膜的融合。在此过程中，细胞能够根据需要释放激素和神经递质。 该研究考察了其中两种关键的融合诱导分子及其相互关系。 这项工作使本科生和研究生能够在生物物理化学领域接受培训，掌握观察细胞行为的尖端技术。 在整个项目中，研究和教育被纳入丹佛公立学校学生的外联活动和丹佛大学非科学专业的教育。这项工作的科学成果在科学会议上和通过出版物传播，以确保将新发现的知识纳入大脑和神经系统细胞功能的更大图景中。膜融合的过程需要能量的输入以将囊泡脂质双层与质膜的脂质双层合并。在这样做的过程中，膜迅速从相对平坦的双层转变为负弯曲的管以形成融合孔。尽管SNARE蛋白驱动融合，但假设某些脂质有助于负弯曲孔的形成。磷脂酸（PA）是一种在体外形成负弯曲膜的脂质，其形成被认为发生在融合部位以稳定膜形状变化。有证据表明，抑制产生PA的酶，磷脂酶D1，减少了质膜上PA的量，膜融合被阻断。尽管已知PA的存在显著影响胞吐作用，但尚不清楚PA在融合位点形成和积累的时间。使用高分辨率的荧光成像方法，PA和磷脂酶D1的位置在分泌过程中被及时绘制。本研究结合哺乳动物细胞培养使用生化重建研究来表征PA如何被招募到融合位点以及PA如何影响膜曲率。最终，研究结果确定了PA如何促进胞吐作用，并导致对细胞中脂质分选的分子理解。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.