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. 2018 Nov 28;8:17447. doi: 10.1038/s41598-018-35593-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Both CLIP-170 phosphorylation and T cell stimulation are essential for MTOC repositioning and full activation of T cells. (a–g) A phosphodeficient S312A mutant of CLIP-170 impairs MTOC repositioning and full activation, and a phosphomimetic S312D mutant does not. Dual-colour epi-fluorescence live-cell imaging (a,b,d and e) at the contact surface in stimulated Jurkat cells using combinations of TagRFP-T-MAP4 (red, bottom) with C-terminal mEGFP-tagged proteins (green, top): wild-type CLIP-170 in the presence of 20 μM compound C (a), phosphomimetic S312D mutant in the presence of 20 μM compound C (b), wild-type CLIP-170 (d) and phosphodeficient S312A mutant (e). MTOC centreing (c,f) in Jurkat cells expressing wild-type- or S312D-mEGFP in the presence of 20 μM compound C (c) and wild-type or S312A-mEGFP (f). The relative expression of IL-2 (g) in Jurkat cells expressing wild-type or S312A CLIP-170. (h–j) MTOC centreing requires phosphorylated CLIP-170 at Ser-312, while MTOC distance from the surface is caused solely by stimulation. TIRF live-cell imaging of S312A-TagRFP-T (h) at the contact surface (top) and by epi-fluorescence at the plane containing MTOC (bottom) in stimulated Jurkat cells. MTOC distance from the surface (i), and MTOC centreing (j), quantified using the images. In panels i and j, the data in Fig. 1c and d are re-plotted for comparison. Bars, 5 μm. Data are means ± SD. Source data, Tables S1 and S3.