Figure 10.
Figure 10. TCR-pMHC microclusters have a reduced density of PS even when Lck activity is blocked. (A) B-A8 T cells expressing PS lipid probe Lact-C2-mRFP were treated ±50 µM PP2 and injected into flow cell with HA-DR4/ICAM-1 lipid bilayers. T cells were imaged using TIRFM at 30-s intervals for 5 min. (top row) Control cells show distinct TCR-pMHC microclusters (green), correlated with reduced Lact-C2 signal in these microclusters (red). (bottom rows) PP2-treated cells show larger pMHC-TCR clusters (green), again correlated with reduction in Lact-C2 signal in these clusters (red). Images representative of n = 3 experiments. Bars, 10 µm. (B) Pearson’s correlation graph of bottom cell in A treated with PP2. (C) Population analysis of at least 20 cells per group assessed for all tested lipid probes including the PP2 treatment experiment in A. Pearson’s correlation factor was calculated by plotting fluorescence signals from lipid probes against HA-DR4 signal using a ROI encompassing only the synapse; correlation factors were plotted as bar graph. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI analysis (*, P = 0.357). n > 20 cells per group. (D) B-A8 T cells expressing CD3ε-YF-TFP were treated with 50 µM PP2 and imaged by TIRFM on HA-DR4/ICAM-1 bilayers. Images representative of n = 3 experiments. Bar, 10 µm. (E) B-A8 T cells that expressed Lact-C2-mRFP, R-pre-mRFP, or MyrPalm-eGFP were imaged for FRAP analysis at the plasma membrane of resting or HA-DR4/ICAM-1 lipid bead–stimulated cells. FRAP was performed at site of synapse for stimulated cells. An area of membrane opposite to FRAP region was selected for normalization. Images are representative for each condition. Region of interest (ROI) is depicted as dotted circle. Images on the right show fluorescence intensities as pseudo-color gradient. Images representative of n = 3 experiments. Bars, 5 µm. (F) FRAP during course of image acquisition (50 s). Results are shown as ratio between fluorescence level of opposing membrane and ROI. Error bars indicate SEM. n = 10 cells per experiment. (G) Bar graph representation of FRAP at 50 s after photobleaching (PB). Error bars indicate SEM. NS, P = 0.5634 (not significant); **, P = 0.0004; ***, P = 0.0290. n = 10 cells per experiment.

TCR-pMHC microclusters have a reduced density of PS even when Lck activity is blocked. (A) B-A8 T cells expressing PS lipid probe Lact-C2-mRFP were treated ±50 µM PP2 and injected into flow cell with HA-DR4/ICAM-1 lipid bilayers. T cells were imaged using TIRFM at 30-s intervals for 5 min. (top row) Control cells show distinct TCR-pMHC microclusters (green), correlated with reduced Lact-C2 signal in these microclusters (red). (bottom rows) PP2-treated cells show larger pMHC-TCR clusters (green), again correlated with reduction in Lact-C2 signal in these clusters (red). Images representative of n = 3 experiments. Bars, 10 µm. (B) Pearson’s correlation graph of bottom cell in A treated with PP2. (C) Population analysis of at least 20 cells per group assessed for all tested lipid probes including the PP2 treatment experiment in A. Pearson’s correlation factor was calculated by plotting fluorescence signals from lipid probes against HA-DR4 signal using a ROI encompassing only the synapse; correlation factors were plotted as bar graph. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI analysis (*, P = 0.357). n > 20 cells per group. (D) B-A8 T cells expressing CD3ε-YF-TFP were treated with 50 µM PP2 and imaged by TIRFM on HA-DR4/ICAM-1 bilayers. Images representative of n = 3 experiments. Bar, 10 µm. (E) B-A8 T cells that expressed Lact-C2-mRFP, R-pre-mRFP, or MyrPalm-eGFP were imaged for FRAP analysis at the plasma membrane of resting or HA-DR4/ICAM-1 lipid bead–stimulated cells. FRAP was performed at site of synapse for stimulated cells. An area of membrane opposite to FRAP region was selected for normalization. Images are representative for each condition. Region of interest (ROI) is depicted as dotted circle. Images on the right show fluorescence intensities as pseudo-color gradient. Images representative of n = 3 experiments. Bars, 5 µm. (F) FRAP during course of image acquisition (50 s). Results are shown as ratio between fluorescence level of opposing membrane and ROI. Error bars indicate SEM. n = 10 cells per experiment. (G) Bar graph representation of FRAP at 50 s after photobleaching (PB). Error bars indicate SEM. NS, P = 0.5634 (not significant); **, P = 0.0004; ***, P = 0.0290. n = 10 cells per experiment.

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