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

Figure 1.

Figure 1

Gp41 and TCR closely associate at the PM of CD4+ T cells. (A) Two-colour PALM imaging of fixed E6.1 Jurkat cells expressing TCRζ-Dronpa and gp41(JRFL)ΔED*-PAmCherry (simply notated gp4-PAmCherry below) on an αCD45-coated coverslips. Cells were dropped and let spread on the coverslip for 3 min before fixation. Shown is a representative cell (N = 20). Bars – 2 μm (left) and 200 nm (right). (B) PCF of TCRζ-Dronpa (green) and gp41-PAmCherry (red). (C) EOM of TCRζ-Dronpa and gp41(JRFL)ΔED*-PAmCherry. (D) Two-colour PALM imaging of fixed E6.1 Jurkat cells expressing TCRζ-Dronpa and gp41(JRFL)ΔED*-PAmCherry on a TCR stimulating, αCD3-coated coverslips for 3 min before fixation. (N = 13). (E) PCF of TCRζ-Dronpa (green) and gp41(JRFL)ΔED*-PAmCherry (red) on stimulatory coverslips. (F) EOM of TCRζ-Dronpa and gp41(JRFL)ΔED*-PAmCherry. Error bars are SEM. (G) (top row) FRET imaging of fixed E6.1 Jurkat cells expressing gp41(JRFL)ΔED*-PAGFP on an αCD45-coated coverslips. TCRs were stained using αCD3ε-Alexa647 primary antibody. Gp41(JRFL)ΔED*-PAGFP molecules were stained using αGFP-Alexa555. Cells were dropped and let spread on the coverslip for 3 min before fixation. Shown is a representative cell (N = 14). Bars – 2 μm. (middle row) FRET imaging of fixed E6.1 Jurkat cells on an αCD45-coated coverslips, as a negative control to the results in the upper row. TCRs were stained using αCD3ε-Alexa647 primary antibody. CD11 molecules were stained using an αCD11-Alexa555 primary antibody. Cells were dropped and let spread on the coverslip for 3 min before fixation. Shown is a representative cell (N = 12). Bars – 2 μm. (bottom row) FRET imaging of fixed E6.1 Jurkat cells on an αCD45-coated coverslips, as a positive control to the results in the top row. TCRs were stained using αCD3ε-Alexa647 primary antibody and a secondary antibody carrying Alexa555 that targeted the αCD3ε-Alexa647 primary antibody. Cells were dropped and let spread on the coverslip for 3 min before fixation. Shown is a representative cell (N = 13). Bars – 2 μm. Colour bar – FRET efficiency.