Extracellular Vesicles: Biogenesis, composition, and biological function.

细胞外囊泡：生物发生、组成和生物功能。

• 批准号: RGPIN-2020-04641
• 负责人: Williams, Karla
• 金额: $ 2.19万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716331
• 关键词: Extracellular; Vesicles; Biogenesis; composition; biological

The study of small lipid enclosed vesicles released by cells, termed extracellular vesicles (EVs), is a rapidly expanding field. The biogenesis of EVs has been documented in virtually every living organism and cell type. EVs were originally thought to be trash containers' of waste and excess products that cells no longer need. It is now well recognized that EVs are important mediators of cell-cell communication and have diverse and active roles in numerous cellular processes. In addition, recently a new non-lipid enclosed small particle termed exomere has been described, adding to the complexity of small particle analysis. Identifying and characterizing proteins involved in EV and exomere biogenesis and function has been challenging. No single technology has been shown to be capable of achieving a complete spectrum of EV properties (size distribution, number, phenotypic EV analysis) in biological samples which limits our knowledge of their functional heterogeneity. A better understanding of EV biology and function will be supported through new technologies capable of estimating size and identifying protein content simultaneously. The protein and nucleic acid content of EVs has been well studied, however, in stark contrast to this is the limited studies assessing the glycan composition of EVs. Studies aimed at uncovering EV-glycan content and function will greatly enhance our understanding of EV biology. Our work aims to make improvements in EV biology by: 1) Development of a nanoscale flow cytometry platform for small particle analysis. Multiple EV and exomere isolation techniques will be used (ultracentrifugation, size exclusion chromatography) and we will generate knockout cell lines with impaired (sub)population release. Our nanoscale flow cytometry platform will be developed to establish size ranges, quantitate EVs, and detect multiple cell surface proteins to phenotype (sub)populations. Validation will performed through Western blot, electron microscopy, and nanoparticle tracking analysis. We will then use this platform to discover regulators of EV and exomere biogenesis or secretion using a bioactive' compound library. 2) Characterize and determine the effects glycosylation, specifically polysialylation, on EV function. We have identified a glycan, polysialic acid (polySia), enriched on cell derived EVs. Our work will assess the route of polySia-EV release and determine its function. We will monitor polySia-EV release in cell culture using live-cell imaging and through knockout studies we will validate the pathway of polySia-EV release. Isolated polySia-EVs will also be used for functional studies. The Williams lab research program long-term goal is aimed at characterizing EV biogenesis, composition, and function to improve our understanding of EV biology. In addition, the Williams Lab is focused on the training of HQP in the field of EV biology. The proposed program will support two graduate and six undergraduate students.

对细胞释放的小脂质封闭囊泡（称为细胞外囊泡（EV））的研究是一个快速扩展的领域。几乎所有生物体和细胞类型中都记录了 EV 的生物发生。电动汽车最初被认为是细胞不再需要的废物和多余产品的垃圾箱。现在人们普遍认识到 EV 是细胞间通讯的重要介质，并且在许多细胞过程中发挥着多样化和积极的作用。此外，最近描述了一种新的非脂质封闭小颗粒，称为外显子，增加了小颗粒分析的复杂性。识别和表征参与 EV 和外显子生物发生和功能的蛋白质一直具有挑战性。目前还没有任何一种技术能够在生物样品中实现完整的 EV 特性（尺寸分布、数量、表型 EV 分析），这限制了我们对其功能异质性的了解。通过能够同时估计大小和识别蛋白质含量的新技术，将支持更好地了解 EV 生物学和功能。 EV 的蛋白质和核酸含量已得到充分研究，然而，与此形成鲜明对比的是评估 EV 聚糖组成的研究有限。旨在揭示 EV 聚糖内容和功能的研究将极大地增强我们对 EV 生物学的理解。我们的工作旨在通过以下方式改进 EV 生物学： 1）开发用于小颗粒分析的纳米级流式细胞术平台。将使用多种 EV 和外显子分离技术（超速离心、尺寸排阻色谱），我们将生成（亚）群体释放受损的敲除细胞系。我们的纳米级流式细胞术平台将被开发用于确定尺寸范围、定量 EV 并检测多种细胞表面蛋白以表型（亚）群。将通过蛋白质印迹、电子显微镜和纳米颗粒跟踪分析进行验证。 然后，我们将利用该平台利用生物活性化合物库来发现 EV 和外显子生物合成或分泌的调节因子。 2) 表征并确定糖基化（特别是多唾液酸化）对 EV 功能的影响。我们已经鉴定出一种聚糖，即聚唾液酸 (polySia)，它富含细胞衍生的 EV。我们的工作将评估 polySia-EV 的释放途径并确定其功能。我们将使用活细胞成像监测细胞培养物中polySia-EV的释放，并通过敲除研究我们将验证polySia-EV释放的途径。孤立的 PolySia-EV 也将用于功能研究。 威廉姆斯实验室研究计划的长期目标是表征 EV 的生物发生、组成和功能，以提高我们对 EV 生物学的理解。此外，威廉姆斯实验室还专注于EV生物学领域HQP的培训。拟议的计划将支持两名研究生和六名本科生。