Extracellular Vesicles: Biogenesis, composition, and biological function.

细胞释放的脂质小囊泡，称为细胞外囊泡（EVs），是一个迅速发展的领域。电动汽车的生物起源已经被记录在几乎每一个生物体和细胞类型。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“垃圾桶”。现在人们已经认识到，ev是细胞间通讯的重要介质，在许多细胞过程中具有多种活跃的作用。此外，最近一种新的非脂质封闭的小颗粒被称为外显子，增加了小颗粒分析的复杂性。鉴定和表征参与EV和外显子的生物发生和功能的蛋白质一直具有挑战性。没有一种技术被证明能够在生物样品中实现EV特性的完整谱（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，将更好地了解EV的生物学和功能。电动汽车的蛋白质和核酸含量已经得到了很好的研究，然而，与此形成鲜明对比的是，评估电动汽车聚糖组成的研究有限。旨在揭示EV聚糖含量和功能的研究将大大提高我们对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仪平台。将使用多种EV和外显子分离技术（超离心，大小排除层析），我们将产生具有受损（亚）群体释放的敲除细胞系。我们将开发纳米级流式细胞仪平台，以建立大小范围，定量ev，并检测多种细胞表面蛋白表型（亚）群体。验证将通过Western blot，电子显微镜和纳米颗粒跟踪分析进行。然后，我们将使用该平台使用“生物活性”化合物文库发现EV和外显子生物发生或分泌的调节因子。2)表征并确定糖基化，特别是多唾液基化对EV功能的影响。我们已经鉴定了一种多糖，聚唾液酸（polySia），富集于细胞来源的ev中。我们的工作将评估polySia-EV的释放途径并确定其功能。我们将利用活细胞成像技术监测polySia-EV在细胞培养中的释放，并通过基因敲除研究验证polySia-EV的释放途径。分离的polysia - ev也将用于功能研究。威廉姆斯实验室研究项目的长期目标是描述电动汽车的生物发生、组成和功能，以提高我们对电动汽车生物学的理解。此外，Williams实验室还专注于EV生物学领域的HQP培训。该计划将资助两名研究生和六名本科生。

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.