Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment.
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 zeta 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.
Institut de Cancérologie et d'Immunologie de Marseille, Université de la Méditerranée, INSERM, Unite 119, Marseille, France.
Grégory
G
Bonello
Nicolas
N
Blanchard
Maria C
MC
Montoya
Enrique
E
Aguado
Claire
C
Langlet
Hai-Tao
HT
He
Selena
S
Nunez-Cruz
Marie
M
Malissen
Francisco
F
Sanchez-Madrid
Daniel
D
Olive
Claire
C
Hivroz
Yves
Y
Collette
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C110810:LAT protein, human; C491734:Lat protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D004247:DNA