The role of LAT clusters in early immune cell signalling [thesis]

David Williamson
T cell activation is an essential part of the immune response and requires segregation of signalling proteins into specialised membrane domains to transmit and sustain T-cell receptor (TCR) signalling. Engaged TCRs initiate signalling through an extended protein network around the adaptor protein Linker for Activation of T cells (LAT). TCR-LAT networks are critical for membrane segregation, sustained signalling and actin cytoskeleton remodeling although the driving forces and interplay of
more » ... ents are not well understood. LAT exists in clusters at the cell membrane before and after T cell signalling. LAT clusters have been associated with ordered membrane domains (lipid rafts) and the actin cytoskeleton, raising questions on the nature and influence these processes may have on LAT signalling during T cell activation. In this study it is demonstrated that the activation of T cells increased membrane order at the T cell activation site, observed using the polarity-sensitive membrane dye Laurdan. The increase in membrane order was independent of LAT cross-linking at a distal site, achieved by specific biotinylation of LAT and adsorption onto streptavidin-beads. LAT clustering induced in this manner also increased local membrane condensation at the bead and was independent of TCR triggering. This suggests that both TCR triggering and LAT cross-linking contribute to membrane re-organisation and the formation of raft domains. High resolution single molecule imaging of LAT and phosphorylated LAT in the plasma membrane of T cells and HeLa cells was conducted using photo-activatable localisation microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). Local point pattern analyses were used to quantitatively map LAT clustering. In HeLa cells, the clustering behaviour of the non-signalling LAT(YAllF) mutant was found to be similar to that of wildtype LAT. The palmitoylation-deficient LAT(C26/29S) mutant formed larger clusters containing more LAT molecules. Conditions of reduced lipid raft abunda [...]
doi:10.26190/unsworks/15153 fatcat:z6dlpwc3qbgphkkhj6blxoey4y