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靶细胞的信号来相互交流。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的反应至关重要。包括癌症、糖尿病和神经退行性疾病在内的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并激活改变细胞行为的级联事件的蛋白质。最近，被称为外泌体的分泌性膜结合囊泡已被确定为一种替代且更复杂的通信模式。它们携带信号及其受体，以及细胞内信号蛋白和RNA。它们的多方面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，以及作为可能的载体将生物活性分子传递给患者体内的缺陷细胞，吸引了人们的广泛关注。外泌体被认为是在细胞内膜结合的“多泡”隔室中形成的，这些隔室被认为起源于称为晚期内体的细胞器。似乎存在多种外泌体亚型，但已证明很难通过实验将它们彼此分离并与其他分泌囊泡分离。因此，不同类型的外泌体的调节和功能仍然知之甚少。不同基因的功能在果蝇中比在哺乳动物中更容易测试。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来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有关的问题。我们在果蝇中鉴定出一种具有巨大的细胞内内体和分泌隔室的特定细胞，并证明了与目前的教条相反，外泌体除了晚期内体之外还在几个不同的隔室中形成。我们已经发现了选择性标记每种外泌体的蛋白质，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，这些新类型的外泌体之一也是在人类癌细胞中产生的。当细胞受到不利条件时，这些外泌体会分泌出来，它们具有特殊的特性，可能有助于肿瘤适应其环境。在这里，我们建议在我们的果蝇系统中完全复制细胞产生的不同外泌体。我们将阻断我们认为可能控制这些外泌体的多个基因的功能，以确定特定的外泌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，阻止选定的外泌体亚型的形成，以确定它们的货物和功能，并确定我们已经确定的控制机制中哪一个是从苍蝇到人类的保守机制。这将使我们能够弄清楚不同类型的外泌体的作用以及外泌体信号如何改变以影响周围细胞的行为。随着对分析外泌体在健康和疾病中的功能以及将外泌体工程化为新的治疗递送系统的巨大兴趣，迫切需要确定存在哪些不同类型的外泌体以及它们是如何制造的。该提案的发现将立即为全球许多研究人员建立一个新的框架，以定义分离物中不同的外泌体亚型，然后有可能选择性地分离它们或阻断它们的分泌。它们还可以提供对外来体及其生物发生机制的新方法的见解，这些方法可以在其他领域进行研究或利用，例如生殖生物学，传染病和害虫控制，其中细胞间和生物体间的通信起着关键作用。

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.