Optimal stress fiber and focal adhesion assembly requires α2β1 integrin and GPVI ligation. (A) MKs preincubated with function-blocking antibody directed against α2β1 integrin (6F1; 10 μg/mL) were plated on collagen I in serum-free medium (i). Cells were allowed to attach and spread for 2 hours before fixation and staining for actin cytoskeleton (rhodamine-phalloidin, red) or vinculin (FITC antibody, green). Serial images were captured starting from the basal level of the adherent cell. Sections are indicated on the right bottom of each image. Note that stress fiber and focal adhesion formation is completely impaired by pretreatment with anti-α2β1 mAb in all examined sections of a representative cell (compare with Figure 1A). Bar equals 10 μm. In panel Aii, the data shown are the mean values ± SD of 300 cells showing stress fibers in 3 independent experiments after treatment by blocking antibodies α2β1 (6F1; 10 μg/mL), monovalent GPVI Fab (clone 9O12.2; 20 μg/mL), or the isotype-matched control (20 μg/mL) and adhesion to collagen I or fibrinogen matrices. (B) Time course of stress fiber and focal adhesion organization after attachment of primary human MKs to slides coated by GFOGER peptide (20 μg/mL), the specific substrate of α2β1 integrin. In each panel, a projection of representative horizontal section of cells stained by TRITC-phalloidin and vinculin (i-iii) showing lamellipodia (arrowheads) and stress fibers (arrows). In panel Biv, MKs were allowed to adhere for 2 hours on CVX. Horizontal confocal section of a projection gradually rotated by an angle of 5° shows the colocalization of actin (red and yellow) and vinculin (green and yellow) in filopodia (arrow) and the attachment plan (arrowhead) in a representative cell (x-y projection view). In panel Bv, the mean ± SD of 300 cells adherent to the indicated substrates for 2 hours and showing stress fibers in 3 independent experiments is shown.