Fibrillar collagen I induces stress fiber and focal adhesion assembly and Rho GTP activation in primary human MKs. (A) MKs were grown for 10 days in the presence of TPO. Cells were then plated onto collagen or PLL-coated coverslips in serum-free medium and incubated for 2 hours. Adherent cells were fixed, permeabilized, and stained with rhodamine-phalloidin (i,iv) and monoclonal antivinculin FITC antibody (ii,v), as described in “Materials and methods.” Photomicrographs were taken with a Zeiss Axiovert confocal microscope (× 60). The cells shown in each panel are representative of predominant morphologies observed in 4 separate experiments. Lamellipodium and stress fibers (i,iii) are observed at the periphery of spread cells (arrowhead and arrow, respectively). Vinculin-rich focal adhesions (indicated by an asterisk) colocalize with the extremities of stress fibers (iii, arrow) in the first basal section (0.8 μm). MKs adherent to PLL substrate display mainly actin aggregates (iv,vi) and diffuse vinculin staining (v,vi) without any substantial spreading. Bar equals 10 μm. (B) The graph illustrates the mean ± SD of adherent MKs showing stress fibers on collagen I or PLL substrates for the indicated times. At least 300 cells were counted in 8 to 10 different fields in 4 separate experiments. (C) Levels of GTP-loaded Rho in primary human MKs kept in suspension or allowed to attach to collagen I-coated dishes for the indicated times. GTP-loaded form of Rho was recovered by precipitation with GST-RBD beads. Non hydrolyzable form of GTP (GTPγS) and guanosine diphosphate (GDP) were loaded as positive and negative controls, respectively. The precipitates (top panel) and total lysates (bottom panel) were subjected to SDS-PAGE and immunoblotting with polyclonal anti-Rho antibody.