Regulation of exosome heterogeneity and function

在所有动物中，细胞通过释放影响附近和远处目标细胞的信号来相互通信。这些信号对于确保所有组织以协调的方式发育并对环境做出适当反应至关重要。癌症、糖尿病和神经退行性疾病等疾病可能涉及这些过程中的缺陷。几十年来，我们已经知道，许多信号是与受体结合的蛋白质，并激活一系列事件，从而改变细胞的行为。最近，被称为外体的分泌性膜结合囊泡已被确定为一种替代的、更复杂的通讯方式。它们携带信号及其受体，以及细胞内的信号蛋白和RNA。它们多方面的信号活动使它们能够完全重新编程细胞行为。正因为如此，它们作为潜在的疾病标志物和信使，以及可能的向患者缺陷细胞运送生物活性分子的载体，引起了人们的极大关注。外体被认为是由称为晚期内小体的细胞器起源的，在细胞内膜结合的多囊体内形成。似乎存在多种外体亚型，但事实证明，很难在实验上将它们彼此分开，以及将其他分泌的囊泡分开。因此，不同类别的外切体的调节和功能仍然不太清楚。我们在果蝇黑腹果蝇身上研究了这个问题。不同基因的功能在苍蝇身上比在哺乳动物身上更容易测试。我们的团队和许多其他研究人员发现，人类和苍蝇的基本细胞机制非常相似，这使我们能够利用苍蝇回答生物学中的基本问题，然后将这些发现应用于研究与动物和人类健康相关的问题。我们在果蝇中发现了一种特殊的细胞，它具有巨大的细胞内内体和分泌室，并证明了与当前的教条相反，除了晚期内体外，外体还在几个不同的室中形成。我们已经发现了选择性地标记每种类型的外切体的蛋白质，并且已经有证据表明，这些亚型的分泌可以独立控制。最值得注意的是，其中一类新的外切体也是在人类癌细胞中制造的。这些外切体是在细胞受到不利条件影响时分泌的，它们具有特殊的性质，可能有助于肿瘤适应它们的环境。在这里，我们建议完全描述由我们的苍蝇系统中的细胞制造的不同外切体。我们将阻断我们认为可能控制这些外切体的多个基因的功能，以找出特定的外切体亚型是如何形成和分泌的。然后，我们将把我们的研究扩展到人类细胞，阻止选定的外体亚型的形成，以确定它们的货物和功能，并确定我们已经确定的哪些控制机制从苍蝇到人类是保守的。这将使我们能够弄清楚不同类型的外切体起什么作用，以及如何改变外切体信号来影响周围细胞的行为。随着人们对分析外切体在健康和疾病中的功能以及将外切体设计为治疗新的输送系统非常感兴趣，迫切需要确定存在哪些不同类型的外切体以及它们是如何制造的。这一提议的发现将立即为世界各地的许多研究人员建立一个新的框架，以定义分离株中不同的外切体亚型，然后有可能选择性地分离它们或阻止它们的分泌。它们还可能为在生殖生物学、传染病和虫害防治等细胞-细胞和生物间交流发挥关键作用的其他领域研究或利用外体及其生物发生机制的新方法提供见解。

In all animals, cells communicate with each other by releasing signals that affect nearby and distant target cells. These signals are vital to ensure all tissues develop in a co-ordinated way, and respond appropriately to the environment. Diseases including cancer, diabetes and neurodegenerative disorders can involve defects in these processes. We have known for decades that many signals are proteins that bind to receptors and activate a cascade of events that changes a cell's behaviour. More recently, secreted membrane-bound vesicles called exosomes have been identified as an alternative and more complex mode of communication. They carry signals and their receptors, as well as intracellular signalling proteins and RNAs. Their multifaceted signalling activity allows them to completely reprogramme cell behaviours. Because of this, they have attracted much attention as potential markers and messengers of disease, and as possible vehicles to deliver bioactive molecules to defective cells in patients.Exosomes are proposed to form inside intracellular membrane-bound 'multivesicular' compartments that are thought to originate from organelles called late endosomes. Multiple exosome subtypes seem to exist, but it has proved difficult to experimentally separate them from each other and other secreted vesicles. The regulation and functions of different classes of exosome have therefore remained poorly understood.We have investigated this problem in the fruit fly, Drosophila melanogaster. The functions of different genes can be much more readily tested in flies than in mammals. Our groups and many other researchers have found that basic cellular mechanisms are remarkably similar in humans and flies, allowing us to use flies to answer fundamental questions in biology and then apply the findings to investigate problems relating to animal and human health. We identified a specific cell in flies that has huge intracellular endosomal and secretory compartments, and have demonstrated that contrary to current dogma, exosomes are formed in several different compartments in addition to late endosomes. We have discovered proteins that selectively mark each type of exosome and already have evidence that secretion of these subtypes can be independently controlled. Most notably, one of these new classes of exosome is also made in human cancer cells. These exosomes are secreted when cells are subjected to adverse conditions and they have specialised properties that may help tumours to adapt to their environment.Here we propose to fully characterise the different exosomes made by cells in our fly system. We will block the function of multiple genes that we think may control these exosomes to work out how specific exosome subtypes are formed and secreted. We will then extend our studies into human cells, blocking formation of selected exosome subtypes to identify their cargos and functions, and determining which of the control mechanisms we have identified is conserved from flies to humans. This will allow us to work out what the different types of exosome do and how exosome signalling can be changed to influence the behaviour of surrounding cells.With huge interest in analysing exosome function in health and disease, and in engineering exosomes as new delivery systems for therapeutics, there is an urgent need to determine what different types of exosome exist and how they are made. Findings from this proposal will immediately establish a new framework for many researchers worldwide to define different exosome subtypes in isolates and then potentially isolate them selectively or block their secretion. They may also provide insights into novel ways in which exosomes and their biogenesis mechanisms could be studied or exploited in other areas, such as reproductive biology, infectious disease and pest control, where cell-cell and inter-organism communication play critical roles.