Elucidate exosome transfer between cells and determine the role of caspases in the regulation of exosome assembly and secretion

建立一种新的生物学模型来阐明细胞间的外泌体转移，并确定在生理条件下半胱氨酸天冬氨酸蛋白酶在外泌体组装和分泌中的作用。外泌体是细胞外囊泡的一种亚型，可以有效地在细胞间转运蛋白质、脂质和RNA。虽然它们在细胞通讯中的工具作用被广泛接受，但负责它们的装载和细胞释放的基本分子机制在很大程度上仍然未知。我们正在开发一个Cre-LoxP报告系统，适用于调查外泌体加载和果蝇S2细胞和苍蝇组织之间的转移。在基于细胞培养的测定中使用该系统的初步结果表明，Cre mRNA可以潜在地包装在细胞外囊泡内并在细胞之间转移。有趣的是，通过添加25个核苷酸的细胞外囊泡“zipcode”，可以实质上增强特定的外泌体加载和随后的mRNA转移。我们的工具目前正在复杂的果蝇组织中进行适应，旨在进一步研究外泌体介导的细胞通讯的生物学。在这方面，我们将把我们的科学努力集中在使用RNA-Seq分析揭示促进外泌体内装载的mRNA的分子特征上。此外，我们将调查的凋亡因子称为半胱天冬酶在外泌体释放的参与。最后，我们的目标是利用这些知识，通过使用装载定制货物的外泌体来远程改变细胞特性。我们的项目有可能提高我们对外泌体加载/转移的基本理解，同时为开发生物工具以操纵通过外泌体进行的细胞间通讯铺平道路。BBSRC优先事项涵盖动物研究中的替代，改进和减少（3R）-将Cre-LoxP系统用于研究外泌体加载和转移到果蝇细胞培养和体内模型，减少了利用在小鼠中建立的类似系统的需要。生物科学的技术开发-基于果蝇细胞的和体内模型，以研究外泌体负载的分子机制及其作为分子载体的潜力，为EV和生物研究社区提供新的资源。世界一流的生物科学-使用协作方法和各种生物/生物信息学技术来回答基本的生物学问题。健康生物科学-外泌体与许多疾病有关，因此，了解它们的基本生物学有助于我们了解它们在疾病中的作用。我们的工作为利用外泌体装载、释放和定向转移治疗分子以实现药物递送铺平了道路。

Establishing a novel biological model to elucidate exosome transfer between cells and determine the role of caspases in the regulation of exosome assembly and secretion under physiological conditions.Exosomes are a subtype of extracellular vesicles that efficiently transport proteins, lipids and RNA, between cells. Although their instrumental role during cell communication is broadly accepted, the basic molecular mechanisms responsible for their loading and cellular release remain largely unknown. We are developing a Cre-LoxP reporter system suitable for investigating exosome loading and transfer between Drosophila S2 cells and fly tissues. Preliminary results using this system in cell culture based assays suggest that Cre mRNAs can be potentially packaged within extracellular vesicles and transferred between cells. Interestingly, specific exosome-loading and subsequent transfer of mRNAs can be virtually enhanced, by adding a 25-nucleotide extracellular vesicle 'zipcode'. Our tool is currently in the process of adaptation within complex Drosophila tissues, aiming to investigate further the biology of exosome-mediated cell communication. In this regard, we will focus our scientific efforts on uncovering the molecular features of mRNAs that facilitate the loading within exosomes using RNA-Seq analysis. In addition, we will investigate the involvement of the apoptotic factors known as caspases in exosome release. Finally, we aim to exploit this knowledge to change cellular properties remotely, by using exosomes loaded with customised cargos. Our project holds the potential to improve our fundamental understanding of exosome loading/transfer, whilst paving the way to the development of biological tools to manipulate intercellular communication via exosomes.BBSRC Priorities CoveredThe replacement, refinement and reduction (3Rs) in research using animals - Adapting the Cre-LoxP system for studying exosome loading and transfer to Drosophila cell culture and in vivo models, reducing the need to utilise a comparable system established in mice.Technology development for the biosciences - Drosophila cell based and in vivo models to investigate the molecular mechanisms of exosome loading and their potential as molecule carriers, providing a novel resource to the EV and biological research communities.World-class bioscience - Using a collaborative approach and varied biological/bioinformatic techniques to answer fundamental biological questions.Bioscience for health - Exosomes have been implicated in a number of diseases and thus, understanding their fundamental biology improves our understanding of their role in disease. Our work paves the way to harnessing the ability to utilise exosomes for the loading, release and directed transfer of therapeutic molecules for drug delivery.WUB, ENWW