ICAP-1 is required for cells to adapt cell velocity as function of substrate stiffness independently on the presence of β1 integrin and is involved in β3 integrin FA translocation to the cell center. (A) Osteoblast cells were spread on FN-coated PAA gels of different rigidities (3, 15, and 60 kPa). Cell migration was monitored for 5 h using time-lapse microscopy. The cell velocity was determined by individually tracking 200–300 cells in three independent experiments. ICAP-1 deficient cells displayed a constant migration velocity whatever the substrate rigidity as compared to the β1 integrin^+/+- icap-1^+/+ cells, independently of the presence of β1 integrin highlighting the crucial role of ICAP-1 in rigidity sensing. Error bars represent SD. N ≥ 199 cells. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. (B) Note that the additional deletion of ICAP-1 in cells depleted in β1 integrin induces an increase of β3 integrin FA translocation to the cell center. Quantification of the percentage of FA whose distance to the nearest cell contour point is >12.5% of the average diameter of the cell. Error bars represent SD. N ≥ 208 cells. ****, P value ≤ 0.0001.