Figure 7. Keratinocytes derived from conditional c-Met mutant mice do not rearrange their cytoskeleton at the scratch wound edges in the pres- ence of HGF/SF. Keratinocytes derived from control and conditional c-Met mutant mice were stained 24 h after scratch wounding, with antibodies directed against vinculin (A), with phalloidin (A and D), antibodies directed against VASP (B), paxillin (C), RhoA (D), and γ-tubulin (E). Arrows mark the newly formed focal contacts (A–C) and RhoA at the rear of the cells (D). Arrowheads mark cytoplasmatic and perinuclear localization of RhoA in mutant (Fig. 7 D, right). The dotted line indicates the edges of the wounds. Bars: (A–D) 50 μm; (E) 20 μm. 158 JCB • VOLUME 177 • NUMBER 1 • 2007 Primary keratinocytes were stained with phospho-c-Met anti- body at different stages of scratch wound closure. In the pres- ence of HGF/SF, control cells showed phospho-c-Met staining in a cobblestone manner (Fig. 6 F). Such a staining was associ- ated with the membrane of virtually all cells that were close to the wounds at 24 h, and also with cells in the healed area at 48 and 96 h (Fig. 6 F). In contrast, only few keratinocytes derived from the skin of conditional c-Met mutant mice were initially phospho-c-Met positive (Fig. 6 G, left, arrow). We observed an enrichment of phospho-c-Met–positive cells at the wound edges after 48 h, and after 96 h a large proportion of cells that had closed the scratch wound contained phospho-c-Met (Fig. 6 G, arrows). Thus, residual c-Met–positive cells preferentially par- ticipate in wound closure in vitro and in vivo. We also examined if primary keratinocytes derived from the skin of control and conditional c-Met mutant mice exhibited different properties at the wound edges. The keratinocytes were characterized by immunohistological analysis using antibodies directed against proteins that are important for directed cell mi- gration, such as vinculin, paxillin, and VASP (Mitchison and Cramer, 1996; Rottner et al., 1999, 2001; Raghavan et al., 2003). Keratinocytes derived from control skin in the presence of HGF/SF showed increased numbers of focal adhesions at the wound edges, and these pointed directly toward the wounds (Fig. 7, A–C, left, arrows). Actin stress fibers, which were stained by phalloidin, were also oriented preferentially toward the wounds (Fig. 7 A). Control cells at the wound edges dis- played a preferential location of RhoA staining that is located at the rear of the cells (Fig. 7 D, left, arrows; Nobes and Hall, 1999; Raftopoulou and Hall, 2004). These control cells at the edges of the wound also reoriented their microtubules, which was demonstrated by γ-tubulin staining (Fig. 7 E, left; Yvon et al., 2002). In contrast, keratinocytes derived from the skin of conditional c-Met mutant mice displayed punctuated, cytoplas- matic, and perinuclear staining of RhoA, which is not located at the rear of the cell (Fig. 7 D, right, arrowheads). Keratinocytes from conditional c-Met mutant mice displayed only few focal contacts and stress fibers, and these were not oriented toward the wounds (Fig. 7, A–C, right). We also used the isolated primary keratinocytes from the skin of control and conditional c-Met mutant mice to study signal transduction by molecules that are crucial for cell proliferation and cell migration (Morimoto et al., 1991; Rubin et al., 1991; Hartmann et al., 1994; Ridley et al., 1995; Khwaja et al., 1998). Erk1/2, Akt, Gab1, and PAK1/2 were phosphorylated, and thus activated, in control cells in the presence of HGF/SF and TGFα (Fig. 8, left). In contrast, phosphorylation of these molecules in keratinocytes derived from conditional mutant mice was pronounced in the pres- ence of TGFα, but not of HGF/SF (Fig. 8 A, left; for quantifi- cation see Fig. 8 B). These data demonstrate that HGF/SF and c-Met signaling is important for the activation of mole- cules that control proliferation and migration of primary kera- tinocytes in cell culture. Activation of this signaling system results in reorganization of adhesion and cytoskeleton com- plexes, such as focal adhesions and stress fibers, which allows cells to move into the scratch wounds.