Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment

Abstract

The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated ζ chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.

A role for novel lipid interactions in the dynamic recruitment of SNX27 to the T-cell immune synapse

“…For example, initial recruitment of the membrane adaptor protein LAT (linker for activation of T cells) is mediated by cholesterol-sphingolipid raft domains at the IS. 33 Although we did not study this question further, it is intriguing that SNX27 was also identified as a protein with a putative CRAC (cholesterol recognition amino acid consensus) motif on its PDZ domain, 34 as these motifs can also mediate membrane localization.…”

The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis

“…2 This process ensures a steady supply of TCRs at the IS to sustain signaling for T-cell activation 3 and has been co-opted by other receptors, such as the transferrin receptor (TfR) and the chemokine receptor CXCR4, 4,5 as well as membrane-bound signaling mediators, such as the kinase Lck and the adaptor LAT. 6,7 Receptor recycling is orchestrated by the small GTPases Rab4 and Rab11. 8 Cargo specificity is achieved with the assistance of specific regulators and effectors.…”

Signaling at the immune synapse: vesicular trafficking takes the stage

“…4 Central membrane-associated regulators of TCR signaling, such as the kinase Lck (lymphocyte-specific protein tyrosine kinase) and the adaptor LAT, also exploit recycling to move to the IS. 5,6 This mechanism has been suggested to ensure a long-lasting supply of receptors and signaling mediators at this location, which is essential to sustain signaling for the extended timeframe required for naive T-cell activation. The report by Larghi and collegues provides now evidence of a direct link between exocytic trafficking and TCR signaling at the IS by implicating the SNARE VAMP7 in the mobilization of vesicular subsynaptic LAT molecules close to TCR activation sites, where they become competent to orchestrate downstream signaling.…”

“…8 In addition to being associated with the plasma membrane in the form of pre-existing clusters, LAT molecules also form an intracellular pool which partially colocalizes with transferrin positive recycling endosomes. 6 While both pools are recruited to the IS, with a rapid mobilization of plasma membraneassociated LAT and a more delayed mobilization for vesicular LAT, it is the intracellular pool of LAT that appears to be centrally required for full LATdependent TCR signaling. 6 This is further supported by recent findings showing that LAT molecules associated with subsynaptic vesicles, rather than LAT molecules forming pre-existing domains at the plasma membrane, are phosphorylated following TCR engagement.…”

“…6 While both pools are recruited to the IS, with a rapid mobilization of plasma membraneassociated LAT and a more delayed mobilization for vesicular LAT, it is the intracellular pool of LAT that appears to be centrally required for full LATdependent TCR signaling. 6 This is further supported by recent findings showing that LAT molecules associated with subsynaptic vesicles, rather than LAT molecules forming pre-existing domains at the plasma membrane, are phosphorylated following TCR engagement. 9 Of note, LAT containing vesicles frequently associate with surface microclusters of the adaptors SLP-76 (SH2 domain containing leukocyte protein of 76 kDa) and GADS (Grb2-related adaptor downstream of Shc) , where phosphorylated LAT is prominently represented, 10 suggesting that vesicular LAT is phosphorylated on encounter of SLP-76 microclusters at the plasma membrane.…”