Figure 3.
Figure 3. Apparent formation of micro–adhesion rings and LAT and SLP76 clusters at weak TCR stimulation. (A) T cells expressing Pyk2-GFP were examined on the planar bilayers. The concentration of loading Ag peptide was changed from 10 nM to 10 µM, and Pyk2-GFP rings and TCR-MCs per cell were counted. (B) Concentrations (conc.) of TCR-MCs and Pyk2-rings at the time point of maximum number for each in A are plotted. (A and B) Data are the mean from 12–24 cells for each condition ± SD. (C) The rates of cells that adhered and moved (shaded bar) or adhered and stopped (unshaded bar) are plotted. Data are the mean from 505–773 cells for each condition ± SD. (D) The number of phosphorylated SLP76 (pSLP) clusters per fixed T cells on the planar bilayers was counted. Data are the mean from 10–24 cells for each condition ± SD. (E, left) T cells were loaded with Fluo-4 and Fura-red and analyzed on the planar bilayers. The ratios of fluorescence intensities of Fluo-4 versus Fura-red were plotted over time. Data are the mean from 64–174 cells for each condition ± SD. (right) T cells were stimulated with the glass beads coated with planar bilayer that was loaded with Ag peptides. IL-2 in the culture supernatants from 105 cells at 24 h was measured by ELISA. Data are representative of three independent experiments. (F) T cells expressing ZAP70-GFP, LAT-GFP, or SLP76-GFP were analyzed on the planer bilayer. Representative images of 20–36 cells at the maximum cell spreading time are shown. (G) The cluster numbers of ZAP70-GFP, LAT-GFP, and SLP76-GFP per cell are plotted over time. Data are the mean from 20–36 cells ± SD. (H) The mean intensities of individual clusters generated during the initial 1 min in G are plotted per time. Data are the mean from 163–272 clusters ± SD. *, P < 0.01. a.u., arbitrary units. (I) T cells expressing both ZAP70-Halo and Pxn-GFP (top), ­LAT-Halo and Pyk2-GFP (middle), or SLP76-Halo and Pxn-GFP (bottom) were examined on the planar bilayers with 10 nM Ag. Representative images of 16–36 cells at the time of maximum cell spreading are shown. Bars, 5 µm.

Apparent formation of micro–adhesion rings and LAT and SLP76 clusters at weak TCR stimulation. (A) T cells expressing Pyk2-GFP were examined on the planar bilayers. The concentration of loading Ag peptide was changed from 10 nM to 10 µM, and Pyk2-GFP rings and TCR-MCs per cell were counted. (B) Concentrations (conc.) of TCR-MCs and Pyk2-rings at the time point of maximum number for each in A are plotted. (A and B) Data are the mean from 12–24 cells for each condition ± SD. (C) The rates of cells that adhered and moved (shaded bar) or adhered and stopped (unshaded bar) are plotted. Data are the mean from 505–773 cells for each condition ± SD. (D) The number of phosphorylated SLP76 (pSLP) clusters per fixed T cells on the planar bilayers was counted. Data are the mean from 10–24 cells for each condition ± SD. (E, left) T cells were loaded with Fluo-4 and Fura-red and analyzed on the planar bilayers. The ratios of fluorescence intensities of Fluo-4 versus Fura-red were plotted over time. Data are the mean from 64–174 cells for each condition ± SD. (right) T cells were stimulated with the glass beads coated with planar bilayer that was loaded with Ag peptides. IL-2 in the culture supernatants from 105 cells at 24 h was measured by ELISA. Data are representative of three independent experiments. (F) T cells expressing ZAP70-GFP, LAT-GFP, or SLP76-GFP were analyzed on the planer bilayer. Representative images of 20–36 cells at the maximum cell spreading time are shown. (G) The cluster numbers of ZAP70-GFP, LAT-GFP, and SLP76-GFP per cell are plotted over time. Data are the mean from 20–36 cells ± SD. (H) The mean intensities of individual clusters generated during the initial 1 min in G are plotted per time. Data are the mean from 163–272 clusters ± SD. *, P < 0.01. a.u., arbitrary units. (I) T cells expressing both ZAP70-Halo and Pxn-GFP (top), ­LAT-Halo and Pyk2-GFP (middle), or SLP76-Halo and Pxn-GFP (bottom) were examined on the planar bilayers with 10 nM Ag. Representative images of 16–36 cells at the time of maximum cell spreading are shown. Bars, 5 µm.

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