Dlg1 depletion affects IS but not VS formation in T cells.

<p><b>A. MTOC polarization toward the nascent IS in Dlg1+ and Dlg1- Jurkat T cells.</b> Left panel: IS conjugates were formed between Jurkat and APC (Raji B) cells. Phosphotyrosine accumulation at the interface between two contacting cells served as marker of a productive IS contact that led to TCR activation and to activation of the downstream signaling cascade. Centrin was visualized simultaneously to localize the MTOC. A polarized MTOC in a Dlg1+ T cell and an unpolarized MTOC in a Dlg1- T cell are seen. The nuclei of Jurkat T cells are shown in blue, the MTOCs are shown in red and indicated by a white arrow. The phosphotyrosine (PTYR) is shown in magenta. The results shown were obtained with the pHIV-H1shRNADlg1 and pHIV-H1shRNACtl vectors expressing the GFP. Similar results were obtained with cells transduced with the lentivirus vectors pHIV-H1shRNADlg1 and Mission shRNADlg1. Right panel: <b>Quantification of MTOC polarization</b> observed in Dlg1+ (n = 131) and Dlg1- (n = 151) T cells in four independent experiments. P = 0.029. <b>B. Cellular localization of Gag in the presence and absence of Dlg1.</b> Dlg1+ and Dlg1- Jurkat T cells were immunostained for Gag and for Dlg1. White arrows indicate Gag accumulation at the VS. The images shown are representative of three independent experiments. <b>C. VS formation in Dlg1+ and Dlg1- Jurkat T cells.</b> Upper panel: HIV-1-infected Dlg1+ and Dlg1- Jurkat T cells were mixed with an equal number of non-infected primary CD4+ target cells and were allowed to establish contacts on poly-L-lysine-coated coverslips for 1 h at 37°C. The cells were fixed, permeabilized, immunostained for Gag and CD4 and analyzed by confocal microscopy. Monosynapses were defined as contacts between a single effector and a single target cell presenting co-localization (in white) of Gag on the infected cell and of CD4 on the CD4+ target cell at sites of contact. Polysynapses were defined as contacts between a single effector cell and multiple target cells. Lower panel: Quantification of cellular contacts, monosynapses and polysynapses between HIV-1-infected Dlg1+ or Dlg1- Jurkat T cells and CD4+ target cells. A total of 1603 Dlg1+ control T cells and 1616 Dlg1- T cells were counted. The data are the means of 3 independent experiments. P = 0.7 for contacts. P = 1.0 for monosynapses. P = 1.0 for polysynapses. Error bars represent SEM. ns = no statistically significant difference. *, P<0.05. Scale bar = 5 µm.