Figure 6.

Directional migration is impaired in Cav1^−/− MEFs and can be rescued by WT Cav1 and p190RhoGAP knockdown, but not by Cav1Y14F. WT and Cav1^−/− MEFs plated on Fn were recorded in random migration by time-lapse video microscopy during a 10-h period (8-min frame interval). (a) Instantaneous velocities of 580–600 cells of each type were quantified and plotted over time on Fn. Cell densities of both populations were equivalent (not depicted). (b) Histograms represent the mean velocities over the 90–330-min period, at which time steady-state velocities were reached. (c) White lines show representative migration tracks of the cell lines indicated at the top of each panel. Composite migration figures were created by coping randomly selected individual migration paths and combining them into a single figure to avoid empty spaces (Pankov et al., 2005). (d) The ID was quantified with Matlab and MetaMorph software. Histograms display the percentage of cells with ID > 0.3 in each cell line. This value (0.3) was the highest mean ID displayed in all experimental conditions tested, and therefore the percentage of cells with ID > 0.3 represents highly directional migratory cells. Data represent means ± SEM based on six independent experiments. n = 300–500 cells of each line. (e) Transwell filters coated with Fn were used to measure the chemotactic response of WT MEFs, Cav1^−/− MEFs, and Cav1^−/− MEFs reconstituted with caveolin-1 or caveolin-1 Y14F using serum as a stimulus. Cells that had migrated during 4 h to the lower surface of the filters were counted in five random fields. Means ± SD from four independent experiments are shown. ^*, Statistically significant versus WT MEFs; ^#, statistically significant versus Cav1^−/− MEFs.