Nature Immunology
3, 1090 - 1096 (2002)
Published online: 21 October 2002; | doi:10.1038/ni847
Imaging antigen-induced PI3K activation in T cellsJulie Harriague
& Georges BismuthWeb Movie 1. (avi 4.6M)
GFP-Akt-PH translocation in T cell-APC conjugates. L625.7 cells were plated on glass coverslips mounted on Petri dishes and incubated overnight at 37 °C with antigen [1  g/ml tt(830-843) peptide] before the addition of GFP-Akt-PH-expressing T8.1 cells previously loaded with Fura-2/AM. Transmitted light, fluorescence and intracellular calcium were measured every 10 s for 20 min. Transmitted light images are superimposed on fluorescence images showing Akt distribution. The black arrow indicates the conjugate shown in Fig. 1. Insert, left side, calcium response in the same conjugates. Web Movie 2. (avi 1.7M)
Inhibition of Akt membrane relocalization by wortmannin. T8.1 cells were incubated with Fura-2/AM and 100 nM wortmannin at 37 °C for 20 min before being added to antigen-pulsed L625.7 cells. Transmitted light images are superimposed on calcium (left) and fluorescence images (right). Images were acquired every 10 sec for 18 min. Web Movie 3. (avi 1.5M)
Delocalization of Akt in a T cell-APC conjugate after PI3K inhibition. T8.1 cells were incubated with Fura-2/AM and added to L625.7 cells pulsed with antigen. Then, 7 min after the beginning of acquisition, 1 ml of buffer containing 100 nM wortmannin was added (+WTN). Transmitted light images are superimposed on calcium images (left) and fluorescence images (right). Images were acquired every 10 sec for 16 min. Web Movie 4. (avi 1.8M)
GFP-Akt-PH translocation in human T cells interacting with DCs. DCs were plated on polylysine-coated glass coverslips in the presence of 0.1 g/ml SEE. Human primary T cells, transiently transfected with GFP-Akt-PH, were added. Left, transmitted light images; right, distribution of Akt. Images were acquired every 10 s for 17 min.
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