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. 2018 Jun 27;8:9747. doi: 10.1038/s41598-018-28114-5

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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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Gp41 promotes TCR phosphorylation and correlates with localized TCR activation. (A) Three-colour diffraction limited microscopy of fixed E6.1 Jurkat cells expressing either TCRζ-Dronpa alone or with gp41(JRFL)ΔED*-PAmCherry or gp41(HXB2)-PAmCherry and stained for pTCRζ on an αCD3-coated coverslips. Cells were dropped and let spread on the coverslip for 3 min before fixation. Bars – 2 μm (left) and 200 nm (right). Shown are representative cells (No gp41, N = 19; Gp41(JRFL)ΔED*, N = 16; gp41(HXB2), N = 18). (B) The total intensity of phosphoTCRζ (pTCRζ) detected at the footprint of individual cells in the samples shown in panel A. (C) The total intensity of pTCRζ detected at the footprint of individual cells in the samples shown in panel A. Mean values and SEMs are shown for each sample type in panels A and B. (D) Three-colour SMLM imaging of fixed E6.1 Jurkat cells expressing TCRζ-Dronpa and Gp41(JRFL)ΔED*-PAmCherry (via PALM) and stained and imaged for phosphoTCRζ (pTCRζ) (dSTORM) on an αCD3-coated coverslips. Cells were dropped and let spread on the coverslip for 3 min before fixation. Bars – 2 μm (left) and 200 nm (right). In zoom, arrows point to representative clusters of high colocalization between gp41(JRFL)ΔED*-PAmCherry and pTCRζ. Shown is a representative cell (N = 9). (E). (left) A scheme for the analysis of phosphorylation enhancement of TCRζ by gp41. First, we selected TCRζ (green) molecules by their proximity to pTCRζ (blue) molecules (selected molecules are marked with filled circles while unselected molecules are marked with empty circles). We then compared the bivariate PCF of these proximity-selected TCRζ (green) molecules with gp41 (red) molecules to the interaction of the same number of randomly selected TCRζ (green) molecules to the gp41 (red) molecules. (right) The inferred scheme of molecular interaction, according to the statistics on left. The red arrow indicates that gp41 correlates with (or promotes) enhanced phosphorylation of TCRζ (green, proximal to blue). The dotted gray arrow marks that TCRζ molecules have an equal probability of being phosphorylated, regardless of their interaction with gp41. (F) Conditional standardized BPCF (SBPCF) of gp41(JRFL)ΔED*-PAmCherry (red) with a TCRζ-Dronpa (green) subpopulation that was selected based on its proximity to phosphorylated TCRζ see main text and analyses section of the SI for further details).