Defining the functional significance of ABC transporters in intercellular communication with the immune system
Principal Supervisor: Professor Andrew DevittLink opens in a new window
Co-supervisor: Dr Alice Rothnie; Dr Ivana Milic
PhD project title: Defining the functional significance of ABC transporters in intercellular communication with the immune system
University of Registration: University of Aston
Project outline:
As cells die in vivo they are removed by healthy ‘undertaker’ cells (phagocytes) in a process that resolves inflammation and prevents disease. This is essential to homeostasis and healthy ageing. For clearance of dying (apoptotic) cells to be efficient and timely, dying cells release extracellular vesicles (EV) to attract phagocytes to sites of cell death1. Crucially, this interaction of EV with the immune system underpins the control of inflammation, a process central to health, regenerative medicine and many important diseases.
Whilst EV are known to be of greatly varying size (<50nm to >1µm) and derived from different compartments of the dying cell, remarkably little is known of the composition of the EV and how this links to function2. Over recent years, our BBSRC-funded work has yielded extensive detail of the EV composition revealing the presence of eicosanoids (small lipid molecules involved in the control of inflammation), exposed phosphatidylserine3 as well the presence of ABC-transporters in the EV membrane which transport eicosanoids and phospholipids. The functional significance of these ABC transporters on EV is unknown.
Working from our extensive existing data and expertise, this project will seek to define the presence and function of these important transporters in the EV-mediated communication of viable and dying cells with the immune system. This novel approach will help define those factors that are essential for EV control of inflammation and shed light on the mechanisms that control regenerative responses. We will address the following key questions. What ABC-transporters are present and active on EV? How does ABC-transporter function impact on the EV composition and function? Do EV of differing sizes and sources interact in a similar manner with the immune system?
The project will use a broad range of techniques to answer these questions including cell culture, particle isolation and analysis, flow cytometry, imaging (light and electron microscopy), assays of immune cell function and inflammation and will harness the power of available ABC-transporter inhibitors.
References:
1Grant, L., Milic, I. & Devitt, A. (2019). Apoptotic cell derived extracellular vesicles: structure-function relationships. Biochemical Society Transactions: 47(2): 509-516.
2Van Niel, G., D’Angelo, G., & Raposo (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews: Molecular Cell Biology: DOI: 10.1038/nrm.2017.125
3Devitt, A., Griffiths, H.R. & Milic I. (2018). Communicating with the dead: lipids, lipid mediators and extracellular vesicles. Biochemical Society Transactions: DOI: 10.1042/BST2016477
BBSRC Strategic Research Priority: Understanding the rules of life - Immunology
Techniques that will be undertaken during the project:
Analysis and interrogation of extensive proteomics data: to identify lead targets for further analysis using systems biology approaches
Cell culture: Isolation of primary cells from human peripheral blood; cell culture of a range of cell lines and primary cells. Mesenchymal stem cell culture to derive viable cell EV involved in regenerative responses.
Vesicle analyses: isolation and analysis of EV structure and function using Exoid tunable resistive pulse sensing, flow cytometry, electron microscopy, analysis of immune-modulating capability in vitro using chemoattraction assays.
Imaging and analysis: Flow cytometry, Western blotting, light and fluorescence microscopy including real-time microscopy and confocal microscopy, automated cell imaging for functional studies
Cell assays: Induction and analysis of cell death, Binding and phagocytosis assays
Mass spectrometry: for the analysis of EV proteome and lipidome