Phospholipase D Regulation of Exosome Secretion

在化学系生命过程化学项目的支持下，丹佛大学的Michelle Knowles正在研究细胞如何控制彼此之间的交流。细胞交流的一种方式是通过分泌称为“外泌体”的纳米级包。外泌体是脂质包裹的容器，携带分子并穿过身体，在那里它们被其他细胞吸收。外泌体可以改变其他细胞的功能，无论是好是坏。 控制细胞如何制造外泌体及其释放的因素尚未完全了解。该奖项支持的研究将集中在脂质修饰蛋白，磷脂酶D（PLD），一种酶，和磷脂酸（PA），这是一种由PLD催化产生的脂质。 PA可以改变膜的形状，并作为一种信号分子。PLD和PA在外泌体的形成及其释放中的作用将被研究。除了产生关于外泌体的新知识外，该项目还将为研究生和本科生创造机会，学习如何使用最先进的技术来解决细胞通讯前沿的问题。 诺尔斯博士和黛娜·洛克（丹佛大学）将为研究生制定一个同伴指导计划。科学和更广泛的影响活动的结果将在会议上提出，并发表，使其他人能够建立在有关细胞调节和学生辅导获得的基础知识。磷脂酶D和磷脂酶A（PLD和PA）可能参与外泌体的形成和/或在释放过程本身。这两个步骤都需要高度弯曲的膜。在本研究项目中，PLD的存在和PA的形成将在外泌体分泌过程中在空间和时间上进行映射。定量荧光显微镜方法将与标准生物化学方法结合使用，以评估外泌体的数量及其从细胞中释放的频率。将在活细胞中评估外泌体生命周期的形成、运输和融合阶段。将改变PLD的活性和存在，以确定何时何地需要PLD和磷脂酸。该项目具有深远的科学影响的潜力;特别是，通过展示生物物理工具/化学生物学的使用，以帮助阐明细胞通信的重要机制。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

With the support of the Chemistry of Life Processes Program in the Division of Chemistry, Michelle Knowles of the University of Denver is studying how cells control their communication with one another. One way that cells communicate is through the secretion of nanoscopic packets called “exosomes.” Exosomes are lipid-coated containers that carry molecules and travel through the body, where they are taken up by other cells. Exosomes can alter the function of other cells, for better or worse. The factors that control how cells make exosomes and their release are not fully understood. The research supported by this award will focus on the lipid-modifying protein, phospholipase D (PLD), an enzyme, and on phosphatidic acid (PA), which is a lipid whose production is catalyzed by PLD. PA can alter the shapes of membranes and acts as a signaling molecule. The role of PLD and PA in the formation of exosomes and their release will be investigated. Beyond generating new knowledge about exosomes, the project will create opportunities for graduate and undergraduate students to learn how to use state of the art technology to address problems at the forefront of cell communication. Drs. Knowles and Dinah Loerke (University of Denver) will develop a peer mentoring program for graduate students. The results of scientific and broader impact activities will be presented at conferences and published to allow others to build upon the foundational knowledge obtained about cellular regulation and student mentorship.Phospholipase D and phospholipase A (PLD and PA) could be involved in the formation of exosomes and/or in the release process itself. Both steps require highly curved membranes. In this research project, the presence of PLD and the formation of PA will be mapped in space and time during exosome secretion. Quantitative fluorescence microscopy approaches will be used in conjunction with standard biochemical methods to assess the number of exosomes and the frequency with which they are released from cells. The formation, trafficking and fusion stages of the exosome lifecycle will be assessed in live cells. The activity and presence of PLD will be altered to determine when and where PLD and phosphatidic acid are required. This project has the potential for far-reaching scientific impact; specifically, by demonstrating the use of biophysical tools/chemical biology to help to elucidate important mechanisms of cellular communication.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.