Effect of Palmitoylation on early T-cell signaling

T-lymphocyte activation relies on the specific molecular recognition of peptide-loaded MHC (pMHC) on an antigen-presenting cell (APC) by the T-cell receptor (TCR). TCR-pMHC binding causes the spatial rearrangement of components of the TCR signaling machinery and the phosphorylation of multiple tyrosines on TCR-associated CD3 by the non-receptor tyrosine kinase Lck. These initial phosphorylation events result in the binding of downstream molecules and ultimately in T-cell activation. Lck is therefore key for TCR triggering and its absence abolishes T-cell activation. Although the sequence of events that leads to T-cell activation has been extensively studied, a detailed mechanistic understanding of how the spatial organization of different signaling components controls their function is still lacking. Notably, Lck contains an N-terminal membrane anchor, which is necessary and sufficient for its membrane targeting as well as a prerequisite for its function. The membrane anchor consists of three lipid modifications: an irreversibly linked myristic acid and two reversibly attached palmitic acid chains. The presence of three lipid modifications appears interesting, since, in principal, myristoylation plus one palmitate chain is sufficient for tight membrane anchorage. Even more so, protein-protein interactions like the association of Lck with the transmembrane co-receptors CD4 or CD8 would actually make acylation dispensable for membrane targeting. Strikingly, CD4 and CD8, as well as other molecules that modulate Lck activity are also palmitoylated. Some of them, like CD4 and CD8, are transmembrane proteins, where the function of palmitoylation is not obvious. At the same time, studies addressing the function of CD4 and CD8 palmitoylation yielded contradictory results. The present project is designed to gain a fundamental understanding of how protein palmitoylation modulates the spatial organization and thereby the functionality of key signaling molecules during early T-cell activation. In the past, the effect of protein palmitoylation has been mostly addressed in biochemical ensemble experiments or classical diffraction-limited light microscopy. In the present proposal, single molecule microscopy and murine primary T-cell model systems are used to get unprecedented molecular insights into the regulation of early T-cell signaling by protein palmitoylation.

During an infection, T cells recognize foreign antigens and mount a specific immune response to eradicate potentially harmful intruders. This recognition process depends on the spatial organization of the T cell plasma membrane. The goal of the present project was to investigate how the organization of specific T cell membrane proteins in so-called nanoclusters is regulated and how it might affect T cell function. In order to detect and characterize nanoclusters, we chose to use single molecule localization microscopy (SMLM) techniques, which are able to optically resolve cellular structures down to ~20 nm radius or less. However, they can also suffer from artefacts that hamper the quantitative interpretation of image data, in particular when it comes to the conclusions on local spatial distributions like nanoclusters. It has been known for some time that SMLM could be affected by such problems. However, no ideal strategy to correct for them had been developed. While the original goal of the present project was to characterize nanoclustering in T cells, initial experiments indicated that published data from the literature, which were the basis for the project, might have underestimated the impact of different sources of artefacts in SMLM. We therefore shifted the focus of the project and developed a novel experimental strategy to detect nanoclustering in SMLM images. We then went on to use our method to apply it on key T cell signaling proteins. Our results showed that nanoclustering is much less common than originally thought. Our work therefore provides a basis to critically revise current models about the mechanisms that lead to T cell activation and adaptive immune responses.