Mechanism of Lck Recruitment to the T-Cell Receptor Cluster as Studied by Single-Molecule-Fluorescence Video Imaging

The T-cell receptor (TCR) signaling has fascinated many researchers, because, in addition to its immunological importance, it appears to provide interesting paradigms for the study of the basic design/concepts on how cellular signaling transduction is carried out and regulated. The multiple signaling pathways triggered by TCR involve various conceptually different and important mechanisms for the regulation of simultaneously working signaling: 2] some signaling pathways have to be activated continuously for a long time (over 24 h); the TCR signaling network should contain circuits to prevent and/or turn off faulty signals ; and it should amplify or reduce the signal when another signal comes from coreceptors, such as CD28 and CD4/8. Tcells achieve such complex signal transduction by recruiting specific signaling molecules to the TCR clusters and their neighborhood, and also by having them escape from the stimulation zone, in the correct order and at the right time; in other words, the recruitment process itself is the fundamental regulation mechanism for this complex signal transduction. Herein, we study the regulation mechanism by which Lck, a Src family kinase, is recruited to the site of TCR clustering. Lck is recruited to the site of TCR clustering during the very early stages after TCR engagement. We observed single molecules of Lck and its N-terminal ten amino acid sequence (N10), both fused with green fluorescent protein (GFP) (Lckand N10-GFP, respectively, Figure 1A) and expressed in the Jurkat T cell line, than 100 m. Such short distances might mark the lower limit of the suggested method for photolithographic patterning. The observed effect of degradation in the dark suggests that dark pores in titanium dioxide particles may have large effect on the photocatalytic performance. As the separation cost of small, highly photoactive titanium dioxide particles, such as the Degussa P-25, is quite high, these results suggest that it may be worth developing relatively large, yet highly porous, titanium dioxide particles, even if these pores are shielded from the impinging UV light.