Lck dynamics as a key part of early T cell signalling

The lymphocyte-specific protein tyrosine kinase (Lck) is a key protein that takes part as the first kinase in the T cell signalling cascade. Upon T cell receptor engagement by an antigen on the surface of an antigen presenting cell, Lck phosphorylates tyrosine residues in cytoplasmic chains of the TCR-CD3 complex. Lck is anchored to the plasma membrane via post-translational modifications and diffuses in the inner leaflet of the plasma membrane. Lck activity is known to be regulated by its conformation and its spatial distribution is linked to its conformational state. Lck diffusion was found to be different in stimulated T cells compared to non-stimulated (resting) cells. However, whether conformational states control the diffusion pattern of Lck in unknown.Here, I utilised a single molecule technique to monitor Lck diffusion patterns and characteristics, namely photoactivated localisation microscopy (PALM) in conjunction with single particle tracking (SPT). Monitoring Lck diffusion via SPT and PALM (sptPALM) enabled imaging single Lck molecules in live T cell and tracking subtle changes in local diffusions. The first part of this work involves validation and optimisation of imaging parameters for Lck single molecule imaging and testing of SPT software via simulations to ensure that the system is appropriate for these experiments. Further, I tested different Lck variants tagged with photoactivatable monomeric cherry (PAmCherry), to check if Lck diffuse in a confined manner when open. The results support the hypothesis that Lck conformation control its temporal redistribution over the membrane by controlling the ability of Lck to interact with other binding partners. Next, I implemented SPT analysis approach that is based on size of confinement zones and angular changes within detected trajectories. The results of the analyses provided evidence for short-lived interaction of kinase-dead Lck with binding partners. Finally, using a FRET biosensor and SPT in two different experimental sets, I showed the influence of a pharmacological compound on Lck redistribution.In summary, this thesis provides evidence of Lck diffusion control via conformational states, that an active kinase domain is responsible for prolonged-stable interaction of Lck with its substrates and that Lck diffusion can be modulated by external intervention by exposure to pharmacological compounds

Conformational states control Lck switching between free and confined diffusion modes in T cells

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.publishe