Regulation of exosome heterogeneity and function

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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.