Kindlin-3–null cells display defective TCR-triggered spreading on ICAM-1 and fail to generate scattered focal dots on immobile ICAM-1 enriched with headpiece-activated LFA-1. (A) Time course of normal and Kindlin-3–null PB T lymphocyte spreading on ICAM-1 (600 sites/μm²) triggered by TCR ligation with soluble OKT3. (B) Time course of normal and Kindlin-3–null T cells spreading on ICAM-1–IgG (600 sites/μm²) or IgG, each co-immobilized with rabbit anti–mouse antibody. Where indicated, OKT3 was immobilized on the rabbit antibody. Values in panels A and B are each the mean ± range of 2 fields of view. **P < .005 (C) Control and Kindlin-3–null T cells spread on the immobilized OKT3/ICAM-1 surface shown in panel B were fixed and stained with Alexa Fluor 568–labeled 327C mAb specific for the active (open) I domain of the LFA-1 β₂ subunit. Scale bar represents 3 μm. Experiments in panels A-C are each representative of 2. (D) Shown is a representative control and Kindlin-3–null cell (taken from panel B, top row) quantitatively analyzed for microclustering by 2D polygon modeling. The number of individual clusters in the indicated cells is shown below the images in the left panels, and the mean number of clusters per cell is shown in the right panel. Eight cells representative of 100 were analyzed.