Fig. 4.

Loss of FGFR1 and FGFR2 reduces velocity and impairs directional migration of cultured keratinocytes. Immortalized keratinocytes from control and K5-R1/R2 mice were subjected to scratch wounding and analyzed by live cell imaging for 13 hours. Eight cells from a minimum of five movies were analyzed for each genotype, including four cells from the front row and four from the fifth row behind the front. All results were reproduced with an independent cell line from control and K5-R1/R2 mice. (A) Parameters analyzed for the quantification of migration. The migrating cells were analyzed for (B) velocity, (C) displacement (linear distance between starting and end point; ‘b’ in A), (D) persistence coefficient (‘b’/‘a’ in A) (Hartwig et al., 2007) and (E) perpendicular movement (‘c’ in A). Bars represent mean ± s.e.m. (F,G) Migrating keratinocytes were stained with an antibody against giantin to label the Golgi apparatus (green) together with rhodamine-conjugated phalloidin (red) and counterstained with Hoechst (blue). Note the polarization of the Golgi apparatus in cells from wild-type mice, but the equal distribution of giantin around the nucleus in cells from K5-R1/R2 mice. Examples of polarized cells are indicated with an arrow, examples of nonpolarized cells are indicated with an asterisk. The scratch is at the top of the pictures. Scale bar: 50 µm. The percentage of cells with polarized Golgi among all cells is shown in G. Bars represent mean ± s.e.m. A minimum of six different pictures from two different cell lines per genotype was analyzed. (H) A representative picture of a migrating cell with a prominent ruffle is shown on the right-hand side. Ruffles are indicated by a white dot. Pictures of a scratch assay experiment were taken to count cells with ruffles. Four time points of the live imaging experiment were chosen. The percentage of cells with ruffles among all cells was determined at the indicated time points. Bars represent mean ± s.e.m.; n≥5.