T-cell interactions with second generation glass-supported lipid bilayers

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One of the key proteins at the heart of the remaining questions on the molecular organization and dynamics of the resting T-cell surface is the Src-type tyrosine kinase, Lck, which initiates signalling in T-cells by phosphorylating the T-cell receptor (TCR). Lck is of considerable intrinsic interest firstly because its activity needs to be constrained in order to prevent uncontrolled T-cell activation, and secondly because it is an unusual surface component insofar as it associates with the membrane via two types of lipid-type anchors i.e. myristoyl and palmitoyl groups, and it is unclear how or whether this affects the distribution and/or function of this kinase. In the case of the TCR, it has been suggested that receptor ligation induces changes in the lipid environment around the receptor, which facilitates the access of downstream signalling molecules, including kinases. A third point of interest is that, in contrast to other Src kinases, Lck associates with the co-receptor CD4, which is an integral membrane protein. It is possible that the lipid micro-environment and dynamics of CD4-bound and -unbound Lck diffusion differ.We propose to characterize the organization and interaction dynamics of a lipid-anchored protein at the T-cell surface, i.e. myristoylated and palmitoylated Lck, both in relation to different lipids, and in comparison to other components of the signalling machinery of the T-cell including CD4, using STED(-FCS) microscopy. We expect these novel experiments to highlight, in thus far unprecedented detail, the organization of key signalling proteins at the T-cell surface, and to create a critical framework for understanding receptor triggering.