TCRζ-Driven Pre-Signaling Organization of Lck in Rab11⁺ Endosomes Shapes TCR Activation

T cell activation relies on the precise spatiotemporal regulation of TCR signaling at the Immunological Synapse, where vesicular trafficking coordinates the delivery of key signaling molecules. While endosomal pools of Lck and its immediate substrate, the TCR{zeta} chain, have been associated with TCR signaling competence, the mechanisms underlying the regulation and synchronization of their trafficking routes remain unresolved. In this study, we simultaneously traced the endosomal localization dynamics of Lck, {zeta} chain and the TCR in unperturbed cells and under conditions that preserve the endosomal network integrity. We identified a previously unrecognized plasma membrane resident pool of {zeta} that exists independently of the TCR complex yet remains competent for phosphorylation and ZAP-70 recruitment. This "standalone" {zeta} population drives the recruitment of Lck and CD45 into Rab11-positive endosomes, establishing pre-assembled, signaling-primed platforms that could provide the means of sustained signaling without requiring continuous receptor engagement. Our findings advance the understanding of how vesicular {zeta} and Lck pools coordinate to shape TCR activation threshold and signaling persistence. Beyond the basic understanding of TCR signaling, these insights hold translational importance for optimizing the design and signaling efficiency of T cell immunotherapies.