Primary Supervisor: Professor Dylan Owen, Department of Immunology and immunotherapy
Secondary supervisor: Dr Maria Makarova
PhD project title: How membrane lipids regulate immune cell function
University of Registration: University of Birmingham
CD4 positive (Helper) T cells are vital white blood cells of the immune system. Their job is to migrate around the body, interrogating other cells for signs of infection and when detected, secrete cytokines to activate other immune cell types. This interrogation occurs at a complex cell-cell junction called the immunological synapse. Here, T cell receptors (TCRs) engage potentially pathogenic foreign peptides and initiate a signalling cascade involving kinases, phosphatases and adaptors, ultimately leading to gene transcription, cytokine secretion and proliferation. Since the early 2000s it has been known that many of the players in proximal T cell signalling – TCR, Lck, LAT, ZAP70 and others are not actually randomly distributed and diffusing on the cell surface but instead for “T cell microclusters”. Until the 2010s, these have been difficult to study because of their small size and the resolution limit of conventional fluorescence microscopy. There are many competing theories about how and why T cell microclusters form, but they seem to be a way of maximising signalling efficiency and avoiding erroneous activation. This project aims to investigate how the lipids in the plasma membrane themselves regulate protein clustering at the immune synapse.
Plasma membrane phospholipids have a diverse range of head groups (size, charge etc) and acyl tail compositions (lengths, saturations etc) – far more than are actually necessary to form a bilayer. This, coupled with the fact that the cell expends energy maintaining lipid diversity (rather than using environmentally available lipids) suggests a regulatory role for lipid diversity. This is further evidenced by the fact that distinct lipids impart different biophysical properties to the bilayer (bending rigidity, fluidity, viscosity and so on) and affect the distribution and diffusion of membrane proteins.
The first part of the project will therefore be to map the T cell lipidome and examine the biophysical characteristics being imposed on the plasma membrane. There are two principal approaches: lipid mass spectrometry applied to whole cell and sub-cellular fractions (e.g. isolated synapses) and advanced fluorescence imaging. Here, we will use probes which label specific lipids and allow the distributions to be mapped, and advanced environmentally sensitive membrane dyes which change their fluorescent properties according to the biophysical and biochemical nature of the bilayer.
The second part will be to manipulate the plasma membrane lipidome. Again, there are several strategies. We will genetically target the lipid trafficking machinery which moves lipids from the ER to the PM. We will use exogenous supplementation of lipids to the growth media, and we will use small molecule inhibitors of enzymes involved in lipid synthesis and metabolisms. Our Aim will be to develop a controllable T cell plasma membrane lipidome.
Finally, we will characterise the effects of this altered (and designed) lipidome on T cells. Firstly, we will again use the most advanced fluorescence microscopy methods available, in this case single-molecule localisation super-resolution microscopy. This method won the Nobel Prize for Chemistry in 2014 and allows the distribution of membrane proteins to be mapped with nanometer precision. We will therefore characterise the effect of the membrane lipidome on membrane protein nanoarchitecture. Finally, we will conduct assays of T cell function – migration studies, measurements of cytokine production and proliferation to measure signalling efficiency through they newly created nanoarchitecture signalling apparatus.
- Williamson, Owen et al, Nature Immunology 2011
- Owen et al, Nature Protocols 2011
- Rubin-Delanchy … and Owen, Nature Methods 2015
BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology
Techniques that will be undertaken during the project:
- Advanced fluorescence microscopy including single-molecule microscopy and tracking, live-cell imaging, multi-spectral imaging and confocal imaging
- Various methods for lipid mass spectrometry
- Cell culture, transfection, immunostaining, western blots
Contact: Professor Dylan Owen, University of Birmingham