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

Lead Research Organisation: Oxford Brookes University
Department Name: Faculty of Health and Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Title 3D EV animation 
Description I coordinated the production of a 3D video animation explaining what extracellular vesicles are and what they can do. We made two versions of the video, one for the general audience (https://www.youtube.com/watch?v=sx6M8UAkUSM) and one for a more scientific audience (https://www.youtube.com/watch?v=OQeRfaQkZCk). The videos can be viewed on a 2D screen but also in 3D using either a virtual reality headset (such as a vive or occulus rift) or cheaper headset. We have used this at several outreach and engagement events and have had a lot of positive feedback. 
Type Of Art Film/Video/Animation 
Year Produced 2018 
Impact The general public and scientific videos have been viewed more than 4,500 times on YouTube alone, and have been featured on blogs and websites (e.g. https://www.exosome-rna.com/extracellular-vesicles-the-cells-secret-messengers/). 
URL https://www.youtube.com/watch?v=sx6M8UAkUSM
 
Description Advising DEFRA (Veterinary Medicines Directorate)
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description MOOC
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
URL https://www.isev.org/page/MOOC
 
Description ExoPop: Extracellular Vesicle Profiling for early detection of leukaemic progression
Amount £100,000 (GBP)
Funding ID C64210/A28052 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2019 
End 05/2019
 
Description Collaboration with Dr Alberto Baena 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided our expertise in EVs to establish a new collaboration to investigate EV transfer and stress response using the model organism Drosophila Melanogaster.
Collaborator Contribution We provided intellectual input and help to supervise two PhD students working directly on this collaboration.
Impact The students are in their second year and have produced excellent preliminary data which are being prepared for a manuscript
Start Year 2017
 
Description Collaboration with Prof Venu Nair 
Organisation The Pirbright Institute
Department Avian viral Diseases
Country United Kingdom 
Sector Private 
PI Contribution We have offered our expertise in EV uptake to investigate whether vesicles released by virally-infected chicken cells can transfer miRNAs to other recipient cells.
Collaborator Contribution Prof Nair contributes his expertise in virology that relates to the poultry industry.
Impact Manuscript in preparation.
Start Year 2017
 
Description Prof Jeremy Simpson 
Organisation University College Dublin
Department UCD Conway Institute of Biomedical annd Biomolecular Research
Country Ireland 
Sector Academic/University 
PI Contribution The project is a BBSRC-SFI funded award to both labs.
Collaborator Contribution My lab is looking at the delivery of extracellular vesicle (EV) cargo and the function of stress EVs, and Prof Simpson is looking at bulk uptake of EVs into cells. In both cases we are running screens to identify genes involved and the mechanisms underlying these processes.
Impact The collaboration is still at an early stage as the project has only been going for 7 months. Thus far we have skyped several times and I have given a seminar at University College Dublin. There aren't any publications at this stage.
Start Year 2017
 
Description EV 3D Video 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I coordinated the production of a 3D video animation explaining what extracellular vesicles are and what they can do. We made two versions of the video, one for the general audience (https://www.youtube.com/watch?v=sx6M8UAkUSM) and one for a more scientific audience (https://www.youtube.com/watch?v=OQeRfaQkZCk). The videos can be viewed on a 2D screen but also in 3D using either a virtual reality headset (such as a vive or occulus rift) or cheaper headset. We have used this at several outreach and engagement events and have had a lot of positive feedback. The general public and scientific videos have been viewed more than 4,500 times on YouTube alone.
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=sx6M8UAkUSM
 
Description Ni Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I was invited to give a talk about my research at the Northern Ireland Science Festival 2019. It was intended for a general audience and, from speaking to audience members afterwards, it had a positive impact on the knowledge and perceptions of people who were present.
Year(s) Of Engagement Activity 2019
URL https://www.nisciencefestival.com/
 
Description Participation in Oxford Brookes Science Bazaar 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We were once again involved in running an EV-themed stand at the science bazaar at Oxford Brookes University (which attracted more than 2,000 people).
Year(s) Of Engagement Activity 2019
 
Description Science Bazaar and Oxford Science Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact We were involved in two public outreach events, the science bazaar at Oxford Brookes University (which attracted more than 2,000 people) and the Oxford Science Week at the Oxford Town Hall which attracted over 1,000 members of the public.
Year(s) Of Engagement Activity 2017
URL https://www.oxscifest.com/