Redistribution of plasma membrane-associated SLP-2 and mitochondria-associated SLP-2 during immunological synapse formation.

<p>Jurkat T cells stably expressing SLP-2-gfp were incubated on planar membranes containing ICAM-1 and anti-CD3 to induce immunosynapse formation. Cells were imaged at early (<5 min) and late (15–30 min) stages of immunosynapse formation to demonstrate SLP-2 redistribution upon TCR ligation. A) Plasma membrane-associated SLP-2 redistribution during immunosynapse formation was imaged by TIRFM, to eliminate signal from the mitochondrial pool of SLP-2. Cell contact with the planar membrane was imaged by IRM, TCR images were obtained by wide-field fluorescence microscopy and the image overlay represents SLP-2-gfp as green and TCR as red. B) Total SLP-2-gfp, including both plasma membrane-associated and mitochondrial pools was imaged at early and late stages of immunosynapse formation. The bright field image shows cells being imaged, IRM images show contact with the bilayer as dark areas, and the TCR, ICAM-1 and SLP-2-gfp fluorescence channels are shown in gray scale and two red-green merges (green is always SLP-2). The dotted lines in the ICAM-1 picture represent, from the periphery to the centre of the picture, the outer boundaries of the distal SMAC, of the pSMAC, and of the cSMAC. Images are representative 3 separate experiments. C) SLP-2-gfp expressing Jurkat cells were transfected with mitochondria-targeted RFP to verify mitochondrial localization of intracellular SLP-2-gfp. Images were obtained by wide-field fluorescence microscopy and are representative of three separate experiments. The dotted lines in the ICAM-1 image are as described in B. The percentage of cells showing segregation of the mitochondrial pool of SLP-2 away from the cSMAC is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037144#pone-0037144-t001" target="_blank">table 1</a> and this organization of SLP-2 into the pSMAC requires anti-CD3 ligation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037144#pone-0037144-t002" target="_blank">table 2</a>).