The results revealed a similar effect of the mutations on the ζ–cytoskeleton interaction (Fig. 2B), as observed when using the MUT cells (Fig. 2A). The CD3ε chain followed the distribution of ζ, as it was not found only in the cytoskeletal fraction of the MUT cells (Fig. 2C). These results suggest that the association between the TCR subunits and the cytoskeleton is mediated via ζ, and that the positively charged ζ motifs are
responsible for this linkage. We further demonstrate that ζ is also associated with actin within cells; while WT ζ is co-immunopercipitated from cell lysates via anti-actin Abs, MUT ζ was undetected (Supporting Information Fig. 5D). Similar results were obtained when comparing the ζ–actin interaction between splenocytes from WT and ζ−D66−150 mice, the latter lacks the two positively charged motifs; only the WT ζ was found associated with actin (Supporting Information Fig. 5D). Together, these find more data indicate a ζ–actin association within T cells, which is mediated via the two positively charged motifs. Next, the role of the cska-TCRs in T-cell activation was assessed. The above-described data showing
that ζ induces actin bundling (Fig. 1F) suggest a structural role for the cska ζ in IS formation/maintenance. To test this possibility, we first analyzed the polar TCR clustering that follows TCR-mediated activation, which is an early step in IS formation. T cells stably expressing WT or MUT ζ were activated with anticlonotypic Ab-coated beads and subjected to immunostaining using anti-CD3ε Abs. Confocal microscopy analyses revealed that the MUT cells were
unable to BGJ398 display polar TCR clustering upon TCR cross-linking, Methocarbamol as opposed to the cells expressing the WT ζ (Fig. 2D). Despite the inability of the MUT cells to display TCR clustering, they could still transmit immediate TCR-mediated signaling events similar to the WT cells, as indicated by the induction of ζ isoforms (phosphorylated and ubquitinated)  and ZAP-70 and LAT phosphorylation kinetics (Supporting Information Fig. 6A–C). Using a more physiological activation condition in which peptide loaded APCs were incubated with WT and MUT T cells expressing the corresponding specific TCR, revealed similar results; the MUT cells could not display a polar TCR clustering and IS formation (Fig. 2E). These results indicate that cska ζ have a key role in the immediate creation and maintenance of TCR clustering that evolves to IS. We next assessed the significance of the cska-TCRs in the outcome of TCR-mediated activation. It was previously demonstrated that TCRs undergo extensive lysosomal degradation following activation, leading to depletion of surface TCRs and intracellular reservoirs . However, while most studies focused on the non-cska-TCRs, the cska-TCRs were largely neglected. Herein, we demonstrate that both cska-TCRs and non-cska-TCRs undergo a similar degradation process upon TCR-mediated activation (Supporting Information Fig. 7A).