Figure 5
Figure 5. MTOC polarization is prevented by C-term AKAP450-GFP expression. (A) Top panel: Cell conjugates were formed between J77 cells expressing GFP (control) or C-term AKAP450-GFP and SEE-stimulated Raji B cells. Cells were stained for endogenous AKAP450 (red). Bottom panel: Cell conjugates were formed between CH7C17 cells expressing GFP (control) or C-term AKAP450-GFP and HA-stimulated HOM-2 APC. Cells were also stained for endogenous AKAP450. Yellow arrows indicate the positions of immune synapses; blue arrow, position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. Scale bars represent 10 μm. Quantification of MTOC translocation in conjugates formed is shown in the graph. More than 300 conjugates were counted for each condition. Data are the arithmetic mean ± SD of MTOC translocation. *P < .05 compared with J77 or CH7C17 cells transfected with GFP (Student t test). (B) T lymphoblasts were incubated with SEE-pulsed Raji cells loaded with CMAC cell tracker (cyan), fixed, and stained for endogenous AKAP450 (red) as in panel A. Top panel: GFP-expressing T lymphoblasts. Bottom panel: C-term AKAP450-GFP expressing T lymphoblasts. Yellow arrows indicate the positions of immune synapses; blue arrow, position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. The graph shows the quantification of MTOC translocation in conjugates. More than 50 conjugates were counted for each condition. Data are the arithmetic mean ± SD of MTOC translocation. *P < .05 compared with T lymphoblast cells transfected with GFP (Student t test). (C) J77 cells expressing C-term AKA450-GFP (M1) or paxillin-GFP (M2) were incubated with SEE-pulsed Raji B cells loaded with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine cell tracker (red) and time-lapse confocal video microscopy sequences are shown at the time indicated. Images were captured at 37°C. Time is shown in seconds. M indicates movie. Scale bars represent 10 μm.

MTOC polarization is prevented by C-term AKAP450-GFP expression. (A) Top panel: Cell conjugates were formed between J77 cells expressing GFP (control) or C-term AKAP450-GFP and SEE-stimulated Raji B cells. Cells were stained for endogenous AKAP450 (red). Bottom panel: Cell conjugates were formed between CH7C17 cells expressing GFP (control) or C-term AKAP450-GFP and HA-stimulated HOM-2 APC. Cells were also stained for endogenous AKAP450. Yellow arrows indicate the positions of immune synapses; blue arrow, position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. Scale bars represent 10 μm. Quantification of MTOC translocation in conjugates formed is shown in the graph. More than 300 conjugates were counted for each condition. Data are the arithmetic mean ± SD of MTOC translocation. *P < .05 compared with J77 or CH7C17 cells transfected with GFP (Student t test). (B) T lymphoblasts were incubated with SEE-pulsed Raji cells loaded with CMAC cell tracker (cyan), fixed, and stained for endogenous AKAP450 (red) as in panel A. Top panel: GFP-expressing T lymphoblasts. Bottom panel: C-term AKAP450-GFP expressing T lymphoblasts. Yellow arrows indicate the positions of immune synapses; blue arrow, position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. The graph shows the quantification of MTOC translocation in conjugates. More than 50 conjugates were counted for each condition. Data are the arithmetic mean ± SD of MTOC translocation. *P < .05 compared with T lymphoblast cells transfected with GFP (Student t test). (C) J77 cells expressing C-term AKA450-GFP (M1) or paxillin-GFP (M2) were incubated with SEE-pulsed Raji B cells loaded with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine cell tracker (red) and time-lapse confocal video microscopy sequences are shown at the time indicated. Images were captured at 37°C. Time is shown in seconds. M indicates movie. Scale bars represent 10 μm.

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