The Influence Of Plasma Membrane Order On TCR Signalling

Abstract

The signalling events that follow T-cell receptor triggering are mediated by multi-molecular complexes consisting of both membrane-associated and cytosolic proteins. Formation of these complexes is driven by a network of protein-protein and protein-lipid interactions. We study how the selective partitioning of signalling components into separate ordered/raft or disordered phases of the plasma membrane controls the assembly and activity of these signalling complexes.Previous work from our laboratory shows that T cell plasma membrane domains engaged in TCR signalling specifically undergo condensation, and that treatment of Jurkat T-cells with polyunsaturated fatty acids specifically disrupts condensed membrane rafts at TCR activation sites. We have also shown that disruption of membrane order by 7-ketocholesterol leads to reduced recruitment of key TCR signalling components as measured biochemically and by TIRF microscopy of fixed cells. We aim to understand the influence of this biophysical phenomenon on the formation and composition of TCR signalling protein/lipid complexes.To this end, we are currently studying the effects of raft disruption by 7-ketocholesterol and polyunsaturated fatty acids on the kinetic behaviour of TCR signalling components. We employ single molecule real time TIRF microscopy of fluorescently labelled TCR signalling proteins as well as biochemical and proteomic analysis of TCR activation domains upon raft disruption.