Abbreviations used: dSTORM, direct stochastic optical reconstruction microscopy; MAL, myelin and lymphocyte; Syt7, synaptotagmin-7; TIRF, total internal reflection fluorescence. M.-I. Thoulouze and A. Alcover contributed equally to this paper. How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCR subunit, and the

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... LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium-and synaptotagmin-7-dependent fusion of LAT and TCR containing vesicles. Fusion of vesicles containing TCR and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCR, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN- production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions.