Centripetal flow of the actomyosin network regulates valency and affinity of LFA-1. (A) Ex vivo CD4⁺ T cells were conjugated with SEE-pulsed Raji B cells and subjected to the indicated experimental paradigm. After fixation, cells were labeled with TS2/4 and Kim127. (B) Representative conjugates showing integrin localization and activation. The outline of the interacting B cell (asterisk) is indicated with a dotted line. Bar, 5 µm. (C) Corresponding 3D rendering of the IS plane. (D–F) The effects of drug treatments on intensities of total (D), extended (E), and open (F) LFA-1 staining at the IS, each normalized to the untreated control. (G) The effects of drug treatments on maintenance of overall LFA-1 recruitment at the IS. (H and I) The effects of drug treatments on the proportion of LFA-1 in the extended (H) or open (I) conformations are shown based on signal intensity of conformation-specific antibodies, divided by the intensity of antibody recognizing total LFA-1. Data from six independent human donors (color coded in D) are shown; at least 30 conjugates were analyzed per condition for each donor. *, P < 0.05; **, P < 0.01; ***, P < 0.001.