Lateral distribution of the T cell receptor and cholesterol detected by high-resolution secondary ion mass spectrometry (P3370)

Abstract

Abstract The engagement of the T cell antigen receptor (TCR) by a specific peptide-MHC ligand initiates a trans-membrane signaling that activates the T cell. An important event in T cell signaling is the recruitment of signaling proteins to the cell membrane and the assembly of a multi-protein (and lipid) signaling complex. Although it has been suggested that lipid/cholesterol composition plays an important role in the regulation of surface signaling complexes, direct visual evidence on lipid/cholesterol participation in the formation of these complexes is still lacking. Here, we characterize the clustering of TCR within T cell and model membranes by secondary ion mass spectrometry (NanoSIMS) with a spatial resolution of 50 nanometers or better. Quantitative information about the chemical composition of membrane complexes can be obtained through the use of orthogonal isotopic labeling of each signaling molecule. Analysis of nanoSIMS images shows the accumulation of TCRs into 60-150 nm clusters/islands within the membrane of an activating T cell and we further attempt to reveal the composition and lateral distribution of TCR and other membrane components, including cholesterol, within model membranes. Because these membrane structures may be shared with many other cell types, we believe the insights we gain in this system will be generalizable to other examples of cell surface receptor signaling.