Background. The immunological synapse is a specialised cell-cell junction between an antigen presenting cell and a lymphocyte (T-cell) composed of >20 immune receptors that function in a concerted manner (Fig.1A). The clinical importance of the immunological synapse can’t be overstated - dysregulation of the synapse results in cancers, autoimmune diseases and immunodeficiency and it is thus a therapeutic target of great interest.
The exquisite organisation of the synapse into radially concentric compartments, called supramolecular activation clusters (SMACs) has been expertly visualised using microscopy. In the centre of the “bullseye” (Fig.1B), the T-cell receptor interacts with peptide-loaded major histocompatibility complex (pMHC) across a ~15 nm gap, with the functional outcome of this interaction tuned by costimulatory receptors in both cells. Surrounding this compartment, adhesion receptors gather to stabilise the junction, while receptors at the outer edge link the synapse to the cytoskeleton. In this manner, the synapse spatially regulates signalling during T-cell activation.
The mechanisms that drive the organisation of the immunological synapse are poorly understood. How do twenty receptors get sorted to three compartments rapidly and efficiently? How are particular receptors excluded from compartments? It is known that formation of the synapse requires extremely fast rearrangement of the membrane and cytoskeleton, with mechanical forces spreading through the membrane via proteins and lipids. Lipid microdomains, formed by lipid-lipid phase separation to create highly ordered “rafts”, have been strongly implicated in synapse formation. This would make sense, as lipid rafts have long been reported to facilitate spatial segregation and concentration of proteins in other biological contexts. Yet enrichment of lipid rafts in the synapse has not been explicitly reported and is the subject of debate.
Interestingly, most synaptic receptors in both the T-cell and the antigen presenting cell have been reported to partition into lipid rafts, undergo clustering or showing differential signalling / binding behaviours in lipid rafts. MHC Class II changes structure and dynamics in lipid microdomains and contains structural features in its transmembrane domains indicative of cholesterol recognition motifs. Despite this body of research, the role of biological membranes in directing sorting/recruitment of proteins to the early synapse remains poorly understood.
Objectives. In this work, we will explore the mechanism of lipid raft partitioning events in the earliest stages of synapse formation starting with two receptors, namely MHC Class II and ICAM-1. These receptors make up the minimal antigen presenting cell requirements for synapse formation in model membranes, and both are reported to localise to lipid rafts, but little is known about the structural impact of raft-partitioning. We will investigate the structures and interactions of these receptors in raft and non-raft membranes to provide a structural description of early protein recruitment events in formation of the immunological synapse.
References:
- Dixon, A.M., Stanley, B.J., Matthews, E.E., Dawson, J.P, Engelman, D.M., Biochemistry, 2006, 45, pp. 5228-5234
- King, G. and Dixon, A.M., BioSyst., 2010, 6, p. 1650-1661.
- Dixon, A.M., Drake, L., Hughes, K.T., Sargent, E., Hunt, D., Harton, J.A., Drake, J.R., Biol. Chem., 2014, 289, 11695-703
- Dixon, A.M. and Roy, S., Human Immunol., 2019, 80, 5-14.
- Lockey, C., Young, H., Brown, J., Dixon, A.M., Biol. Chem. 2022, doi: 10.1016/j.jbc.2022.101843, PMID: 35307351.
BBSRC Strategic Research Priority: Understanding the rules of life – Structural Biology, and Immunology, Sustainable Agriculture and Food - Animal Health and Welfare, and Integrated Understanding of Health - Ageing.
Techniques that will be undertaken during the project:
- Protein expression and purification
- Protein characterisation using mass spectrometry and circular dichroism and fluorescence spectroscopies
- Solution-state NMR spectroscopy
- Protein purification (HPLC, affinity chromatography)
- AlphaFold2-based protein folding.
- Molecular Dynamics (MD) simulations.
- Structural Bioinformatics.
- Python-based programming
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