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. 2017 Aug 21;7:8952. doi: 10.1038/s41598-017-08357-4

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© The Author(s) 2017

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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Arp2/3 complex-promoted branched actin polymerization regulates the polarization of TCR⁺ endosomes during immune synapse formation. (a) Immunofluorescence analysis of CD3ζ (green) and F-actin (red) localization in conjugates of Control or Arpc2 KD Jurkat T cells with SEE-pulsed Raji cells (blue). Bar is 5 μm. (b) The histogram shows the percentage of conjugates that harbored CD3ζ and F-actin at the immune synapse. At least 30 cells were analyzed for each experiment. (experiment, n = 4). (c) Immunofluorescence analysis of EEA1 (red) and internalized CD3 (green) moved to immune synapse in conjugates pulsed with SEE-loaded Raji B cells (blue). Bar is 5 μm. (d). The histogram shows the percentage of conjugates that harbored EEA1 and recycled CD3 at the immune synapse. At least 30 cells were analyzed for each experiment. (experiment, n = 3) (e–g) Immunofluorescence analysis of (e) EEA1/ (f) Rab5/ (g) Rab11 (red) and CD3ζ (green) in Control and Arpc2 KD Jurkat T cells in conjugates with SEE-pulsed Raji B cells (blue). Bar is 5 μm. (h–j) The histogram shows the percentage of conjugates harboring (h) EEA1/ (i) Rab5/ (j) Rab11 at the immune synapse. At least 30 cells were analyzed for each experiment. (experiment, n = 3) (k) Immunofluorescence analysis of CD3ζ (green) and F-actin (red) in conjugates of control or cytD treated Jurkat T cells with SEE-pulsed Raji B cells (blue). Bar is 5 μm. (l) The histogram shows the percentage of conjugates that harbored CD3ζ and F-actin at the immune synapse At least 30 cells were analyzed for each experiment. (experiment, n = 4). The data are means ± S.D., for all panels: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by Student’s t-test. All results are representative from at least three independent experiments.