Dynamics of p56lck translocation to the T cell immunological synapse following agonist and antagonist stimulation

To study the spatio/temporal recruitment of lck during immunological synapse formation, we utilize highspeed time-lapse microscopy to visualize green fluorescent protein (GFP) fusions of lck and CD3zeta following agonist or altered peptide ligand (APL) stimulation. The dynamics of lck and CD3zeta recruitment are comparable; however, lck becomes excluded to the periphery of mature synapses, while most CD3zeta is centrally localized, suggesting a limited time frame within which lck can efficiently phosphorylate CD3zeta molecules during synapse maturation. Exposure of T cells to specific APLs affects the efficiency of conjugate formation and lck accumulation. Most surprisingly, we find an intracellular pool of lck associated with recycling endosomes that translocates to mature synapses within 10 min of calcium flux. This bolus of lck may contribute to intermediate-late signal transduction.