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

了解神经传递的进展：胞吐作用中磷脂酶 D 活性的时间映射

• 批准号: 1807455
• 负责人: Michelle Knowles
• 金额: $ 45万
• 依托单位: University of Denver
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1807455&HistoricalAwards=false
• 关键词: Progress; Towards; Understanding; Neurotransmission; Temporal; Progress; Towards; Understanding; Neurotransmission; Temporal

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.

有了这一奖项，化学过程的化学过程计划正在支持丹佛大学的米歇尔·诺尔斯博士的研究，以研究冲动从细胞到细胞的关键方面，就像神经系统中一样。 化学信号被装入“发送”细胞的囊泡中，该囊泡以“接收”细胞的方向排出。 许多分子必须在空间和时间上组织，以触发囊泡与接收细胞膜的融合。这样一来，细胞能够按需释放激素和神经递质。 该研究检查了这些关键融合诱导的分子中的两个及其相互关系。 这项工作使本科生和研究生可以接受生物物理化学领域的培训，他们掌握了尖端技术来观察细胞的行为。 在整个项目中，研究和教育都纳入丹佛公立学校的学生以及丹佛大学的非科学专业的教育中。这项工作的科学结果是在科学会议上和出版物中传播的，以确保将新发现的知识纳入大脑和神经系统中细胞的功能的更大了解。膜融合的过程需要输入能量才能将囊泡脂质双层与质膜融合。这样，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形成负弯曲的孔。磷脂酸（PA）的形成是一种体外形成负弯曲膜的脂质，被认为在融合部位发生以稳定膜形状的变化。有证据表明，抑制使PA，磷脂酶D1的酶减少质膜和膜融合处的PA量。尽管知道PA的存在极大地影响了胞吐作用，但尚不清楚融合部位的PA形成和积累的时机的清晰图像。使用高分辨率，荧光成像方法，在分泌过程中将PA和磷脂酶D1的位置映射。这项研究结合了生化重构研究与哺乳动物细胞培养的结合，以表征PA如何招募到融合位点以及PA如何影响膜曲率。最终，这些发现决定了PA如何促进胞吞作用并导致对细胞中脂质排序的分子理解。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子和更广泛影响的评估来审查标准的评估。