Extracellular Vesicles: Biogenesis, composition, and biological function.

对细胞释放的小脂质封闭囊泡（称为细胞外囊泡（EV））的研究是一个迅速扩展的领域。EV的生物起源几乎在每一种活的生物体和细胞类型中都有记录。电动汽车最初被认为是电池不再需要的废物和多余产品的“垃圾容器”。现在已经充分认识到EV是细胞间通讯的重要介质，并且在许多细胞过程中具有多样且活跃的作用。此外，最近已经描述了一种新的称为exomere的非脂质封闭的小颗粒，增加了小颗粒分析的复杂性。鉴定和表征参与EV和外显子生物发生和功能的蛋白质一直具有挑战性。没有一种技术能够在生物样品中获得完整的EV特性谱（大小分布、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鉴定蛋白质含量的新技术，将支持对EV生物学和功能的更好理解。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聚糖组成的有限研究。旨在揭示EV-聚糖含量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（超离心、尺寸排阻色谱），并且我们将产生具有受损（亚）群体释放的敲除细胞系。我们的纳米级流式细胞仪平台将被开发用于建立大小范围，定量EV，并检测多种细胞表面蛋白质的表型（亚）群体。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，我们将使用这个平台来发现调节EV和外显子生物合成或分泌使用的“生物活性”化合物库。 2)表征并确定糖基化（特别是聚唾液酸化）对EV功能的影响。我们已经鉴定了在细胞衍生的EV上富集的聚糖，聚唾液酸（polySia）。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，并通过敲除研究验证polySia-EV释放的途径。分离的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，威廉姆斯实验室专注于EV生物学领域HQP的培训。拟议的方案将支持两个研究生和六个本科生。

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.