During an immune response, T-cell receptors (TCRs) are engaged by the antigenic peptides bound to major histocompatibility complexes (pMHC) of the antigen presenting cells (APC), forming an immunological synapse. Subsequently, the immunoreceptor tyrosine-based activation motifs (ITAM) on TCR/CD3 complex are phosphorylated by the Src kinase Lck, which is a crucial signaling event requiring the kinase and receptor to co-localize in the same cluster. Understanding the dynamics and formation of these clusters is essential information for the solution of the immunological synapse puzzle.
In order to map the timescale of the dynamic self-association of Lck and measure its conformation-dependent partitioning kinetics, we perform the video rate photo-activation microscopy experiment in live T-cells. The intermittent emission of the fluorescent tags, the high density of activated particles and a relatively low signal-to-noise ratio, makes single particle tracking (SPT) exceedingly difficult. We resolve this problem by applying image correlation spectroscopy (ICS) analysis to the acquired image time series of Lck in Jurkat T-cells. We investigate the difference in the spatio-temporal dynamics and kinetics of Lck and its conformation mutants including Lck-Y505F (open) and Lck-Y394F (closed), in either activated or resting Jurkat T-cells. In order to map the phase space for which ICS analysis is valid, we simulate live PALM data for varying particle diffusion coefficients, signal-to-noise ratio, photo- activation, reversible and irreversible photo-bleaching rates.
In summary, we have developed a new analysis for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.