Lck Cluster Dynamics in Live Cells

Abstract

T-Lymphocytes initiate an adaptive immune response after specific binding of the T-cell receptor (TCR) to an antigenic peptide bound to the major histocompatibility complex (peptide-MHC) on an antigen presenting cell. Key events upon TCR-pMHC binding involve the phosphorylation of intracellular tyrosine residues of the TCR and recruitment of adapter molecules, which results in downstream signaling. Initial TCR phosphorylation is primarily carried out by lymphocyte specific kinase (Lck) making it a central molecule for T-cell signaling. Lck has been shown to form nano-scale clusters that can be visualized by super-resolution microscopy techniques. However, neither the molecular determinants nor the function of Lck clustering are clearly understood. In principal, local lipid heterogeneities of the plasma membrane, actin-driven compartmentalization and protein-protein interactions between clustered molecules could account for non-random distribution of Lck on the plasma membrane. We carried out single molecule fluorescent microscopy of mEOS3.2-tagged Lck expressed in JCaM1.6 cells to elucidate the mechanisms that lead to Lck clustering in the plasma membrane. Our single molecule tracking data show transient immobilization of Lck molecules in clusters of about 150 nm. Strikingly, these clusters appear to coincide with topological protrusions of the membrane as indicated by the increased brightness of clustered Lck molecules as well as a fluorescent membrane probe. We further find that neither disruption of the actin cytoskeleton nor cholesterol depletion influence Lck clustering. Finally, we map the region of Lck responsible for its immobilization to the N-terminal unique domain (SH4) using truncation mutants of Lck.