Figure 5. Cells expressing R1049E are deficient in spreading, adhesion, and migration.

Vinculin null mouse embryo fibroblasts expressing GFP, or a fusion of GFP and wild type vinculin (vinWT) or GFP and R1049E vinculin (vinRE) were examined. (A) Expression levels of the various vinculins and GFP. Total cell lysates were harvested from the indicated cells and blotted with antibodies against vinculin to show the level of re-expressed protein or a loading control (p34-Arc). (B-D) RE expressing cells do not spread as well as WT expressing cells. The indicated cell lines were allowed to spread on fibronectin coated surfaces for 25 minutes (C) or 4 hours (B and D). Representative images of cells spreading at 4 hours and stained with DAPI and phalloidin are shown in B, scale bar =25µm. (C and D) At least 30 cells from each of three independent were analyzed to determine cell area – the means ± SEM are shown. (E) RE cells do not adhere as well as WT cells to fibronectin. Cells were seeded on glass coverslips coated with 10µg/ml fibronectin and allowed to adhere for 25 minutes. The coverslips were washed and adherent cells were counted in nine non-overlapping fields. Fold-adhesion is reported relative to the total number of GFP cells adhered, which is set equal to 1.0. Shown is the mean of at least 3 independent experiments ± SEM. (F) RE cells display a migration speed that is intermediate between WT and GFP cells. Cells were seeded on fibronectin and allowed to adhere overnight. Cells were imaged for a total of 10 hours, and only those cells visible for a minimum of 4 hours were included in the analysis. Cells from at least three independent experiments were tracked using NIH ImageJ Manual Cell Tracker plugin; a minimum of 16 cells total were analyzed. The asterisks represent the following: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; One-way ANOVA with Tukey correction for multiple comparisons.