Figure 1.
Figure 1. TCR-MCs in the early phase of activation are bordered with focal adhesion molecules. (A) T cells were fixed on the planar bilayer, stained with anti–LFA-1–Dy549 and anti-TCR–Alexa Flour 647 Abs, and analyzed with a confocal microscope. Representative images of 58 cells are shown. (B) T cells expressing Pxn-GFP were stained with H57(Fab)-Dy549 (TCR) and analyzed on the planar bilayer by TIRF microscopy. The images at the contact area are shown (top left). For better visualization, the image in the boxed area is shown at higher magnification (bottom left). Representative images of 24 cells are shown. The fluorescent intensity profiles of Pxn-GFP and TCRs between the white arrows are plotted (middle). The pixel intensities of Pxn-GFP and TCRs from 188 pixels in the central area as indicated by the arrows were normalized to their median values and plotted (right). a.u., arbitrary units. (C) T cells expressing both Pyk2-GFP and Pxn-Halo were examined. The representative images of five cells are shown. The fluorescent intensity profiles of Pyk2-GFP and Pxn-Halo between the white arrows are plotted (middle). The pixel intensities of Pyk2-GFP and Pxn-Halo from 188 pixels analyzed the same as in B are plotted (right). (D) Fixed T cells were stained with anti-vinculin–FITC and anti-CD3–Alexa Fluor 647 mAbs and analyzed as in A. Representative images of 58 cells are shown. (E) T cells expressing Pyk2-GFP were similarly analyzed. Sequential images of Pyk2-GFP and TCR are shown in chronological order. Representative cells of 23 cells were selected. (F) Criterion for counting micro–adhesion ring is shown for Pyk2 and TCR images. TCR-MCs surrounded by more than half of their periphery with Pyk2 were counted. In this example, the three images from the top are positive, and the one on the bottom is negative. (G) The images of each individual TCR-MC and Pyk2-GFP ring from the cell shown in E (left) and an array illustrating the duration of 43 TCR-MCs and micro–adhesion rings (right) are shown. Red bars indicate the total lifetime of each TCR-MC, and green bars show the period when it was bordered by the Pyk2-GFP ring indicative of micro–adhesion ring formation. Time 0 means the time point of observed cluster formation. Bars: (A–E) 5 µm; (F and G) 1.5 µm.

TCR-MCs in the early phase of activation are bordered with focal adhesion molecules. (A) T cells were fixed on the planar bilayer, stained with anti–LFA-1–Dy549 and anti-TCR–Alexa Flour 647 Abs, and analyzed with a confocal microscope. Representative images of 58 cells are shown. (B) T cells expressing Pxn-GFP were stained with H57(Fab)-Dy549 (TCR) and analyzed on the planar bilayer by TIRF microscopy. The images at the contact area are shown (top left). For better visualization, the image in the boxed area is shown at higher magnification (bottom left). Representative images of 24 cells are shown. The fluorescent intensity profiles of Pxn-GFP and TCRs between the white arrows are plotted (middle). The pixel intensities of Pxn-GFP and TCRs from 188 pixels in the central area as indicated by the arrows were normalized to their median values and plotted (right). a.u., arbitrary units. (C) T cells expressing both Pyk2-GFP and Pxn-Halo were examined. The representative images of five cells are shown. The fluorescent intensity profiles of Pyk2-GFP and Pxn-Halo between the white arrows are plotted (middle). The pixel intensities of Pyk2-GFP and Pxn-Halo from 188 pixels analyzed the same as in B are plotted (right). (D) Fixed T cells were stained with anti-vinculin–FITC and anti-CD3–Alexa Fluor 647 mAbs and analyzed as in A. Representative images of 58 cells are shown. (E) T cells expressing Pyk2-GFP were similarly analyzed. Sequential images of Pyk2-GFP and TCR are shown in chronological order. Representative cells of 23 cells were selected. (F) Criterion for counting micro–adhesion ring is shown for Pyk2 and TCR images. TCR-MCs surrounded by more than half of their periphery with Pyk2 were counted. In this example, the three images from the top are positive, and the one on the bottom is negative. (G) The images of each individual TCR-MC and Pyk2-GFP ring from the cell shown in E (left) and an array illustrating the duration of 43 TCR-MCs and micro–adhesion rings (right) are shown. Red bars indicate the total lifetime of each TCR-MC, and green bars show the period when it was bordered by the Pyk2-GFP ring indicative of micro–adhesion ring formation. Time 0 means the time point of observed cluster formation. Bars: (A–E) 5 µm; (F and G) 1.5 µm.

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