Elucidating the mechanisms and pathways of extracellular vesicle uptake and intercellular stress response.

Billions of years ago life consisted solely of single-celled organisms; these types of creatures tend to compete with one another, and their primary goal is to grow and reproduce. When multi-cellular organisms evolved they had to solve a problem: how to stop individual cells in the organism from fighting and competing with each other, and to actually work together for the benefit of the organism.
One way that organisms solved this problem was by getting cells to communicate with one another in different ways. This communication between cells is crucially important, as it allows them to coordinate important decisions, such as when to grow and when to die. Understanding this communication is therefore a requisite for understanding how multicellular life is regulated.

One of the ways in which cells communicate is via the release of extracellular vesicles (EVs). EVs are essentially tiny 'bags' that are released by cells which carry various cellular components such as proteins and RNA molecules (these are a copied version of DNA which act as an intermediary in the formation of proteins). We have known about the existence of EVs for decades, but it was thought that they were essentially a waste disposal system that cells could use to jettison unwanted material. However, it has emerged that EVs are actually very important. After they are released they can be taken up by other cells, where they can induce a response. In other words, they are part of the communication process that cells use to coordinate their function. Scientists across the world took note and began testing to see if EVs were involved in their favourite topics of research. In our lab we have found that when cells get stressed they are able to send EVs to their neighbours. These 'bystander cells' appear to become damaged, but they also are now more protected against stress. In other words, when cells get stressed they can warn their neighbours to 'toughen up' and prepare some danger heading their way. This seems to play an important role in helping organisms to survive stressful conditions, yet little is known about how EVs control this process.
In this proposal we will aim to better understand this EV mediated communication following stress by pursuing three core objectives:

1. To study the molecular mechanisms of this intercellular stress response

Our preliminary work has revealed some of the molecular steps involved in changing the neighbouring cells to allow this adaptation to stressful conditions. Here we will perform further work to better understand how these steps fit together in controlling the overall effect in the neighbouring cells.

2. To discover genes involved in EV processing.

Nobody has ever comprehensively tested how EVs are able to stick to their target cells, enter those cells and then avoid destruction once inside the cell. Here we will attempt to tackle this difficult but important question. We have designed some experiments which will tell us what proteins are involved in controlling these different steps. The design of the experiments will also allow us to find out what genes are involved in controlling the stress response induced by EVs.

Objective 3 - To study genes identified in objective 2 in more detail.

This will allow us to better understand the mechanisms by which stressed cells are able to communicate with one another. Indeed, we will be able to characterise the whole process from the arrival of the EVs at the bystander cell, the uptake and processing of the EV and the subsequent induction of a response that helps that cell to prepare for future danger. The findings will also broadly appeal to scientists working in a range of different topics.

Extracellular vesicles (EVs) are a type of vesicle that is released by cells into the extracellular space. There has been a recent renaissance in the study of EVs which has coincided with the realisation that they are not simply routes of extracellular waste disposal as had previously been believed. Indeed, it is now widely acknowledged that EVs can play an important role in a variety of processes, including angiogenesis, cell motility and immune regulation. Despite the increasing panoply of functions that EVs are involved in no study has ever set about trying to comprehensively identify the proteins involved in mediating their uptake and processing into recipient cells. In addition it is unclear how EVs released by stress can help coordinate intercellular responses to non-physiological conditions. We will address this shortcoming by performing a range of experiments, including genome-wide RNAi screens combined with high-throughput microscopy and flow cytometry to identify proteins involved in EV uptake and stress response.
In addition to measuring uptake, our experimental design will allow us to simultaneously identify proteins involved in the release of EVs and their cargo from endosomal compartments following internalisation as well uncovering whether EVs with different functional effects on cells are taken up by different pathways. We will also be able to assess whether EVs released by stressed cells, which we have shown are qualitatively different and are involved in coordinating a homeostatic intercellular stress response, are taken up by different mechanisms to regular EVs. We will also be able to identify and test the role of different genes in regulating this intercellular stress response mediated by EVs.
Thus, the work will uncover a wide range of information which will be of benefit to a wide range of research and could profoundly influence our understanding of EV-mediated communication.

This project will be the first comprehensive analysis of EV uptake and processing mechanisms in cells. It will also help us to identify genes involved in the mechanisms of intercellular stress response, as well as give insight into how EVs and their cargo can escape the endosome. The work would therefore have a wide impact on the research community. It would lead to collaborations with our group and stimulate a wide variety of further work in the EV, intracellular trafficking and stress response fields. This impact would be further felt by increased competitiveness in these fields within the UK and further funding from research councils and elsewhere. The work could also have impact by improving delivery of biological tools or therapeutic drugs, which could increase the efficiency of research or improve the outlook for patients. Commercialisation of potential applications would also impact on the local and UK economy. Thus there will be a high potential impact to the research community, industry and patients within the UK and beyond.