Micro–adhesion ring formation is dependent on MyoII activity. (A–C) T cells expressing Pxn-Halo or SLP76-Halo were pretreated with 50 µM Blebb for 3 h and examined on the planar bilayers. (A) The numbers of clusters with (black line) and without (gray line) Blebb treatment are plotted over time. Data are the mean from 12–16 cells ± SD (left and middle). The mean concentration of TCR-MCs at the peak of cluster numbers with (black bar) and without (gray bar) Blebb treatment is plotted (right). Ctrl, control. (B) The mean intensities of individual clusters of TCRs or SLP76 generated during the initial 1 min with (black line) and without (gray line) Blebb treatment analyzed in A are plotted. Data are the mean from 416–540 clusters ± SD. *, P < 0.01. a.u., arbitrary units. (C) T cells were fixed at 2 min and stained with anti–MyoII-FITC, anti–LFA-1 (CD11a)–Dy549, and anti-CD3(TCR)–Alexa Fluor 647 (top). For better visualization, the images in the boxed areas are shown at a higher magnification (middle). Representative images of 12 cells are shown. The fluorescence intensity profiles of MyoII-FITC (green) and LFA-1 (CD11a)–Dy549 in between the white arrows in the top image are plotted (bottom left). The percentages of MyoII cluster area colocalized with LFA-1 or with TCRs as a control to the total MyoII cluster area were plotted (bottom right). Bars, 3 µm. Data are the mean from 12 cells ± SD. *, P < 0.01.