Ena/VASP Mislocalization Decreases the Prevalence of Fast, Persistent-Moving Smooth Cells
(A) EGFP-AP4-mito (negative control) binds to mitochondria in the cell body, but does not mislocalize VASP, which, by immunofluorescence, appears as a thin line at the leading edge of cells with a smooth morphology (arrowheads).
(B) EGFP-FP4-mito mislocalizes VASP at the surface of mitochondria thus preventing its function at the leading edge. Weak VASP localization was only observed at the leading edge in two cells out of more than 50 cells examined. Scale bar = 10 μm.
(C) Keratocytes were classified as either having a smooth or rough leading edge within populations of migrating keratocytes expressing the aforementioned EGFP-tagged constructs. Cells with a rough leading-edge morphology are more prevalent than those classified as having a smooth morphology when EGFP-FP4-mito is expressed (70%) compared with controls, EGFP (55%) and EGFP-AP4-mito (59%). The incidence of rough keratocytes is significantly lower (43%) when EGFP-VASP is overexpressed (when compared to EGFP-FP4-mito, p < 0.05).
(D) Keratocytes exhibiting smooth leading edges are significantly faster than those with rough leading-edge morphology (EGFP, p = 0.0002; EGFP-AP4-mito, p = 0.0004; EGFP-FP4-mito, p = 0.0004; EGFP-VASP, p = 0.0065). Mean and SD are plotted.
(E) To perform qualitative comparisons of keratocyte turning during migration, trajectories of smooth or rough keratocytes were reoriented to start at x,y = 0 in the +y direction in standardized coordinate systems (left and middle panels). Trajectories were truncated (x,y limits = 150 μm) for illustration purposes. The distance between tick marks is 50 μm. For quantitative comparisons, mean angles between velocity vectors separated by specific distances traveled by cells were plotted. Larger mean angles correspond to increased curvature in the trajectories of migrating cells (right panels). Mean angles are significantly smaller for smooth keratocytes expressing negative control constructs compared to rough cells (EGFP, p = 0.0003; EGFP-AP4-mito, p = 0.001), showing that smooth keratocytes maintained straighter paths than rough ones. Ena/VASP protein mislocalization using EGFP-FP4-mito causes smooth cells to move in more curved trajectories with larger mean angles not significantly different from those of rough cells and significantly different from those of smooth cells expressing control constructs (compared to EGFP smooth, p = 0.01; EGFP-AP4-mito smooth, p = 0.005). On the other hand, when keratocytes overexpress EGFP-VASP, rough keratocytes, which have increased trajectory curvature in controls, have straighter paths with smaller angles similar to those from smooth cells expressing controls and EGFP-VASP. Mean and SEM are plotted.