Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism

Abstract

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of four proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute to both pathways, are in our cellular context, specifically and respectively, involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point to the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

Localization in vesicles, clusters and supramolecular complexes as key elements of LAT function

“…Using imaging approaches, it is estimated that 30-75% of the cellular pool of LAT resides in vesicles that are clearly separate from the plasma membrane [5][6][7]. Additional vesicles close to or docked at the plasma membrane are unlikely to be included in this quantification as they are within the diffraction limit of detection in light microscopy.…”

Characterization of endogenous Kv1.3 channel isoforms in T cells

“…Raji B cells (1 × 10 6 cells/mL) were labeled with CellTracker Blue CMAC dye (10 µM, Thermo Fisher) in RPMI without FCS for 20 min at 37°C and incubated with 100 ng/mL SEE for 30 min at 37°C (Saez et al, 2021). SEE-loaded Raji cells and Jurkat cells were cocultured on coverslips for 30 min at 37°C at a ratio 1:1, were washed with PBS, fixed with 4% paraformaldehyde, quenched with 10 mM glycine for 10 min, and permeabilized with staining buffer (PBS + 0.2% BSA + 0.05% saponin) for 30 min.…”

F-Actin Dynamics in the Regulation of Endosomal Recycling and Immune Synapse Assembly

“…Among these are the uncoordinated 119 protein (Unc119), which extracts PM-bound Lck by sequestering its hydrophobic myristoyl group and releases the kinase at the synaptic membrane under the control of the ARL3/ARL13B complex ( Stephen et al, 2018 ); the membrane protein MAL that, together with the formin INF2, generates specific carriers for Lck targeting to the IS in a Cdc42-Rac1-dependent manner ( Andrés-Delgado et al, 2010 ; Antón et al, 2011 ); and the Rab11 effector FIP3 (Rab11 family interacting protein-3), which plays a key role in the regulation of the subcellular localization and function of Lck ( Bouchet et al, 2017 ). LAT trafficking to the IS occurs through the classical Rab5 and Rab11 route, where anterograde transport is specifically regulated by the golgin GMAP210, the intraflagellar transport protein IFT20 and the SNARE VAMP7 and retrograde transport by Rab6 and SNARE Syntaxin-16 ( Larghi et al, 2013 ; Vivar et al, 2016 ; Carpier et al, 2018 ; Zucchetti et al, 2019 ; Saez et al, 2021 ).…”