Molecular Basis and Regulatory Mechanisms of Exosome Secretion

摘要 外泌体是由细胞分泌的小的细胞外囊泡。外泌体携带生物活性分子，如 信号蛋白和mRNA，并有力地影响其受体细胞的病理生理学。资助的项目 通过我的R35资助“外泌体分泌的分子基础和调控机制”（R35 GM 141832）， 了解控制外泌体的生物发生和分泌的分子机制， 通过致癌信号调节外泌体生物发生。由于小尺寸（直径30-120 nm）， 外泌体，纳米粒子跟踪分析（NTA）设备是必不可少的拟议研究。新技术评定 设备执行的功能范围从外泌体数量的定量，测量外泌体直径， 涉及基于荧光的货物标记和外来体亚群鉴定。如果没有这些基本的外泌体 由于这些特性，我们提出的大多数实验都无法进行。在这个建议中，我申请 用于购买ZetaView® x30 dual的设备基金（NOT-GM-22-017）的行政补充 激光多参数NTA设备，以取代我们破碎的设备，NanoSight N300。我们计划购买一个 ZetaView®x30，来自Particle Meetings，Inc.而不是来自Malvin公司的NanoSight NS 300。因为 ZetaView®x30是市场上领先的NTA设备，其功能满足我们的研究需求，而 旧的NanoSight NS 300系统在这些年中暴露出许多缺陷。事实上， 该系统是唯一一种允许我们精确表征外泌体的NTA设备，并且是不可或缺的， 实现R35补助金中提出的目标。

ABSTRACT Exosomes are small extracellular vesicles secreted by cells. Exosomes carry bioactive molecules such as signaling proteins and mRNAs, and potently affect the pathophysiology of their recipient cells. The project funded by my R35 grant “Molecular Basis and Regulatory Mechanisms of Exosome Secretion” (R35 GM141832) aims to understanding the molecular mechanisms that control the biogenesis and secretion of exosomes, and the regulation of exosome biogenesis by oncogenic signaling. Due to the small size (30-120 nm in diameter) of exosomes, a nanoparticle tracking analysis (NTA) equipment is essential for the proposed study. The NTA equipment carries out functions ranging from quantification of exosome number, measuring exosome diameters, to fluorescence-based cargo labeling and exosome sub-population identification. Without these basic exosome characterizations, most of our proposed experiments cannot be carried out.In this proposal, I apply for Administrative Supplements for Equipment fund (NOT-GM-22-017) for the purchase of a ZetaView® x30 dual laser multiple parameter NTA device to replace our broken equipment, NanoSight N300. We plan to purchase a ZetaView®x30 from Particle Metrix, Inc. rather than the NanoSight NS300 from Malvin, Inc. because ZetaView®x30 is a leading NTA equipment in the market with features meeting our research needs, whereas the old NanoSight NS300 system has a number of deficiencies revealed over these years. In fact, ZetaView system is the only NTA device that allow us to characterize exosomes with precision, and is indispensable for the accomplishment of the goals proposed in the parent R35 grant.