Enabling mechanistic studies of EVs in vivo: a protocol for isolation and cell-specific labelling in larval zebrafish.

IF 8.2 2区 生物学 Q1 CELL BIOLOGY
Ezgi Kiyga, Katy Reid, Guillaume van Niel, Julie Mazzolini, Dirk Sieger
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引用次数: 0

Abstract

Background: Extracellular vesicles (EVs) are critical mediators of intercellular communication in development, physiology, and disease. In vivo models such as Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio (zebrafish) now provide powerful platforms to visualize EV dynamics in real time. However, the full potential of these models remains underutilized due to the lack of reliable, cell-specific EV labelling tools and robust EV isolation protocols. Here, we present an optimized workflow for the isolation of EVs from zebrafish larvae and the in vivo labelling of EVs in a cell-type-specific manner.

Methods: To isolate EVs from larval zebrafish, we used size exclusion chromatography (SEC). By comparing different tissue digestion methods and performing step-by-step optimisation of sample preparation prior to SEC, we established a novel protocol that enables EV isolation without compromising cell viability. EV size and concentration were assessed by nanoparticle tracking analysis (NTA), with subsequent characterization by transmission electron microscopy (TEM) and Western blotting. To evaluate the sensitivity of our protocol, we treated zebrafish larvae with GW4869, a known inhibitor of EV biogenesis, and assessed the dose-dependent effects on EV release. To specifically label EVs from distinct cell types, we have generated a UAS: CD63-GFP construct which can be expressed under control of the Gal4 transcriptional activator.

Results: Through a systematic comparison of tissue dissociation techniques, we identify Bacillus licheniformis protease as a superior alternative to conventional collagenase treatment, which compromises cell integrity. Treatment with GW4869 confirmed that EV biogenesis and release can be inhibited in a dose-dependent manner and demonstrated that our protocol is sensitive enough to detect and quantify changes in EV levels. To enable cell-specific EV tracking in vivo, we combined the UAS: CD63-GFP construct with a radial glia-specific Gal4 driver line, providing a proof-of-concept for targeted EV imaging in intact tissues.

Conclusions: These advances provide a versatile toolkit for mechanistic studies of EV function in vivo. The broad availability of cell-type-specific Gal4 driver lines in zebrafish and Drosophila will now allow researchers to trace EV dynamics from virtually any cell type, while our isolation protocol enables rigorous, quantitative EV analyses across developmental and pathological contexts.

使ev在体内的机制研究成为可能:在斑马鱼幼虫中分离和细胞特异性标记的方案。
背景:细胞外囊泡(EVs)是发育、生理和疾病过程中细胞间通讯的重要介质。活体模型,如黑腹果蝇、秀丽隐杆线虫和斑马鱼,现在为实时可视化EV动态提供了强大的平台。然而,由于缺乏可靠的细胞特异性EV标记工具和强大的EV分离协议,这些模型的全部潜力仍未得到充分利用。在这里,我们提出了一种优化的工作流程,用于从斑马鱼幼虫中分离ev,并以细胞类型特异性的方式对ev进行体内标记。方法:采用大小排斥色谱法(SEC)从斑马鱼幼虫中分离ev。通过比较不同的组织消化方法,并在SEC前逐步优化样品制备,我们建立了一种新的方案,使EV分离不影响细胞活力。通过纳米颗粒跟踪分析(NTA)评估EV的大小和浓度,随后通过透射电子显微镜(TEM)和免疫印迹(Western blotting)进行表征。为了评估我们的方案的敏感性,我们用GW4869(一种已知的EV生物发生抑制剂)处理斑马鱼幼虫,并评估了对EV释放的剂量依赖性影响。为了特异性标记来自不同细胞类型的ev,我们生成了一个UAS: CD63-GFP结构,该结构可以在Gal4转录激活子的控制下表达。结果:通过对组织分离技术的系统比较,我们确定地衣芽孢杆菌蛋白酶是传统胶原酶治疗的优越选择,这损害了细胞的完整性。用GW4869处理证实了EV的生物发生和释放可以以剂量依赖的方式被抑制,并证明我们的方案足够敏感,可以检测和量化EV水平的变化。为了在体内实现细胞特异性EV跟踪,我们将UAS: CD63-GFP结构与径向胶质细胞特异性Gal4驱动系结合,为完整组织中的靶向EV成像提供了概念验证。结论:这些进展为EV在体内功能的机制研究提供了一个多功能工具箱。斑马鱼和果蝇中细胞类型特异性Gal4驱动系的广泛可用性将使研究人员能够从几乎任何细胞类型中追踪EV动力学,而我们的分离方案能够在发育和病理背景下进行严格、定量的EV分析。
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来源期刊
CiteScore
11.00
自引率
0.00%
发文量
180
期刊介绍: Cell Communication and Signaling (CCS) is a peer-reviewed, open-access scientific journal that focuses on cellular signaling pathways in both normal and pathological conditions. It publishes original research, reviews, and commentaries, welcoming studies that utilize molecular, morphological, biochemical, structural, and cell biology approaches. CCS also encourages interdisciplinary work and innovative models, including in silico, in vitro, and in vivo approaches, to facilitate investigations of cell signaling pathways, networks, and behavior. Starting from January 2019, CCS is proud to announce its affiliation with the International Cell Death Society. The journal now encourages submissions covering all aspects of cell death, including apoptotic and non-apoptotic mechanisms, cell death in model systems, autophagy, clearance of dying cells, and the immunological and pathological consequences of dying cells in the tissue microenvironment.
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