Cell spreading requires the interaction of talin with Tiam1 and integrin. (A) Lysates of COS7 cells expressing Tiam1-C1199-HA were subjected to pull-down assay with GST-fused talin1 mutants. Tiam1 was barely detected in the eluates of beads containing talin1 A366/368. (B) GST-tagged integrin β3 cytoplasmic region (wild type or Y747A) was subjected to pull-down with lysates of COS7 cells expressing myc-talin1 (1–433 aa). Talin1 A366/368 bound to integrin β3, but talin1 A358 did not. (C) U251 cells transfected with talin1 siRNA and the indicated plasmids were stained with anti-GFP and anti-Tiam1 antibodies. Bars, 10 µm. Insets in the leftmost images (merged) are magnified in the panels to the right. The bar graph on the right represents the number of Tiam1-containing adhesions per 100 µm². Wild-type talin1 restored the inhibitory effect of talin1 knockdown on Tiam1, but the talin1 mutant A366/368 did not. (D) U251 cells transfected with the siRNA against talin1 and indicated plasmids were seeded onto FN under serum-free conditions. Rac1 activity was measured by pull-down with PAK-CRIB. Expression of wild-type talin1 rescued Rac1 activation, but that of the talin1 mutants failed to do so. Data represent the means ± SD of three independent experiments. (E) Transfected cells were seeded onto FN-coated dishes and immunostained 2 h later with anti-GFP antibody (green) and phalloidin (red). Bar, 10 µm. The bar graphs below represent quantifications of spread area and cell perimeter. *, P < 0.05; **, P < 0.01 versus respective control cells (Student’s t test). Results are representative of more than three experiments.