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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00968

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

Grégory Bonello^1,*, Nicolas Blanchard^2,*, Maria C. Montoya³, Enrique Aguado⁴, Claire Langlet⁴, Hai-Tao He⁴, Selena Nunez-Cruz⁴, Marie Malissen⁴, Francisco Sanchez-Madrid³, Daniel Olive¹, Claire Hivroz^2,*, and Yves Collette^1,*,,

¹ Institut de Cancérologie et d'Immunologie de Marseille, Université de la Méditerranée, INSERM, Unite 119, Marseille, France
² Institut National de la Santé et de la Recherche Médicale, Unite 520, Institut Curie, Paris, France
³ Servicio de Immunologia, Hospital de la Princesa, Universidad Autonoma de Madrid, Madrid, Spain
⁴ Centre d'Immunologie de Marseille-Luminy, INSERM-CNRS-Université de la Méditerranée, Marseille, France

View larger version (69K): [in a new window]   Fig. 1. Localization of LATWT-, LAT32-104-GFP and LATCt-GFP in T cells. (A) Schematic representation of the mouse LAT constructs. Domains include transmembrane (TM), palmitoylated cysteine residues (CC), green fluorescent protein (GFP) and tyrosine (Y) residues. (B) Jurkat cells expressing LATWT-GFP, LAT32-104-GFP or LATCt-GFP were pulsed with transferrin coupled to cyanine 3 (Tf) and labelled either with the mAb CTR433, which stains the cis-Golgi, or an anti- mAb. Shown are single-colour or two-colour overlay images acquired with a confocal microscope at a medial Z-section. Insets display 3x enlargement of details comprised in the white box. White arrows point to co-localization areas and white arrowheads show vesicles where only Tf is found. Bar, 5 µm. (C) GFP fluorescence intensity of the intracellular and plasma membrane pools of LAT was measured on 12-bit confocal images. Percentage of intracellular versus total LAT is plotted for LATWT-GFP- (, n=29), LATCt-GFP- (, n=33) and LAT32-104-GFP- (, n=28) expressing cells. Shown are mean±s.d., which are significantly different (P<0,0001, unpaired Student's t-test). (D) T-cell blasts were fixed and co-labelled with anti-LAT (left panel) and anti- (right panel) Abs. Images show the presence of endogeneous intracellular pools of LAT and . Bar, 5 µm.

View larger version (49K): [in a new window]   Fig. 2. Tyrosine phosphorylation and raft partition of LAT constructs. (A) LATWT-, LAT32-104- and LATCt-GFP cells were left unstimulated or triggered with anti-CD3 mAb for 2 minutes, and were then lysed and immunoprecipitated with anti-GFP. Immune complexes were sequentially analysed by anti-phosphotyrosine and anti-GFP immunoblotting. (B) Postnuclear supernatants obtained from sonicated LATWT- and LATCt-GFP cells were lysed in Brij 98 at 37°C followed by sucrose gradient fractionation. Raft (L) and non-raft fractions (H) were collected and analysed by anti-GFP immunoblotting. (C) LATWT-, LAT32-104- and LATCt-GFP cells were transfected with the red-shifted GFP (RSGFP) construct. After 36 hours, cells were incubated or not in PBS 1%, Triton X-100 for 15 minutes at 4°C and mounted on slides. RSGFP and GFP staining was analysed by confocal microscopy.

View larger version (90K): [in a new window]   Fig. 3. Role of LAT C-terminal tail in TCR-induced recruitment of LAT to the immune synapse. APCs labelled with the CellTrackerTM Orange were loaded for 20 minutes with 5 µg/ml SEE, mixed with LATWT-, LAT32-104- and LATCt-GFP T cells and monitored by time-lapse confocal microscopy. Images were taken at 10-second intervals. Representative images taken from the digital movies at the times indicated (seconds) are shown. White and black arrowheads point respectively to the initial site of contact and to the intracellular GFP-labelled compartment. Bar, 5 µm.

View larger version (49K): [in a new window]   Fig. 4. Tyrosine residues 136, 175, 195 and 235 are required for TCR-induced recruitment of LAT to the immune synapse. (A) Schematic representation of the mouse LAT and LATY/F GFP chimaeras. (B) LATWT- and LATY/F-GFP cells were left unstimulated or triggered with anti-CD3 mAb for 2 minutes. Whole cell lysates (WCL) and GFP immunoprecipitates (IP) were sequentially analysed by anti-pTyr and anti-GFP immunoblotting. (C) LATWT- and LATY/F-GFP cells were mixed with CellTrackerTM Orange-labelled APCs loaded for 20 minutes with 5 µg/ml SEE and monitored by time-lapse video microscopy. Images were taken at 15-second intervals. Representative images from the digital movies at the indicated times (seconds) are shown. White arrowheads point to the site of initial contact.

View larger version (29K): [in a new window]   Fig. 5. LAT is required for its own TCR-induced recruitment to the immune synapse. (A) Transiently transfected LATWT-, LATCt- and LATY/F-GFP Jurkat cells were added to CellTrackerTM Orange-labelled APCs for 3 minutes. Cells were then fixed and analysed by confocal microscopy. Histograms depict the percentage of T cells, after 3 minutes or 7 minutes of contact with SEE-pulsed APCs, showing either a clear clustering of LATWT-GFP (left panel) or a polarization of the intracellular LAT-containing compartment towards the APC (right panel). Quantification was performed on more than 35 conjugates for each LAT construct. Results of one representative experiment out of two are shown. Bar, 5 µm. (B) GFP fluorescence of LATWT-, LATCt-, LATY/F- and LAT32-104-GFP in JCAM2.5 cells interacting with SEE-pulsed Raji B cells. JCAM2.5 transiently reconstituted with the four LAT constructs were added to CMTMR-labelled (red) Raji B cells and shortly centrifuged to synchronize the contacts. After 8 minutes, cells were fixed and analysed by confocal microscopy. White arrowheads point to the site of initial contact and white stars show the LAT-containing intracellular compartment when visible. (C) Histograms depict the percentage of T cells showing either a clear clustering of LATWT-GFP (grey) or a polarization of the intracellular LAT-containing compartment towards the APC (black). Quantification was performed blindly on 15 conjugates for each LAT construct. Black star indicates the weak intracellular expression of LAT32-104-GFP, such that polarization could not be properly assessed. Bar, 5 µm.

View larger version (12K): [in a new window]   Fig. 6. NF-AT-dependent transcription in LAT-deficient cells reconstituted with different mutants. The NF-AT-Luc plasmid and the indicated LAT constructs were transfected into JCAM2.5 cells. 18 hours after transfection, transfected cells were incubated in standard round-bottom 96-well plates without or with Raji B cells (ratio 0.5 B cell for 1 T cell), either unpulsed or pulsed with 1 µg/ml of SEE. Luciferase activity was assayed 6 hours later using a Promega luciferase assay kit according to the manufacturer's instructions. The percentages and mean fluorescence of GFP-transfected cells were checked by FACS analysis to ensure similar transfection efficiencies. One representative experiment out of three is presented.