Figure 4. Increased VASP phosphorylation in focal adhesions located at the tips of ventral stress fibers.

(A) VASP localizes to focal adhesions at the tips of both dorsal and ventral stress fibers. Focal adhesions positioned at the tips of dorsal (dsf FA) and ventral stress fibers (vsf FA) are indicated by red and orange arrowheads, respectively. Bar, 10 μm. (B) Line profile intensity graphs of the adhesions (highlighted in panel A) and adjacent stress fiber regions show similar localizations of VASP and vinculin in focal adhesions positioned at the tips of dorsal and ventral stress fibers. (C) The domain structure of VASP. Phosphorylation of VASP at Ser239 and Thr278 inhibits its actin polymerization activity. (D) Localization of phospho-Ser239 VASP in focal adhesions at the tips of dorsal (red arrowheads) and ventral stress fibers (orange arrowheads). Bar, 5 μm. (E) Line profile intensities along the yellow lines (indicated in panel D) demonstrating that phospho-Ser239 VASP is enriched at the tips of ventral stress fibers, but not at the tips of dorsal stress fibers. phospho-Ser239-VASP–red; actin-green (F) Quantification of the relative fluorescence intensity ratio of phospho-Ser239-VASP: total VASP in focal adhesions located at the tips of dorsal (dsf FAs) and ventral (vsf FAs) stress fibers. The obtained intensity value from ventral stress fibers was set to 1. Mean intensity values (+/- SEM) of 21 adhesions are shown. (G) Western blot analysis demonstrating that expression of dominant active Rho-14V expression leads to an increase in the total Ser239 phosphorylation levels of VASP. (H) Quantification of the relative phospho-Ser239-VASP: total VASP ratios in control and Rho-14V transfected cells. The ratio in control cells was set to 1 and the mean values (+/- SEM) from three separate experiments are shown. (I) Western blot analysis demonstrating increased phospho-Ser239-VASP levels in cells grown on stiff (64 kPa) martix compared to cells grown on compliant (0.5 kPa) matrix. (J) Quantification of the relative phospho-Ser239-VASP: total VASP ratios in cells grown on soft (0.5 kPa) or rigid (64 kPa) matrices. The phospho-Ser239-VASP: total VASP ratio in cells from stiff matrix was set to 1 and the mean values (+/- SEM) from three separate experiments are shown.

DOI: http://dx.doi.org/10.7554/eLife.06126.016

Figure 4—figure supplement 1. VASP regulates the elongation of dorsal stress fibers.

(A) Depletion of VASP from U2OS cells by siRNA leads to loss of dorsal stress fibers (examples indicated by red arrowheads in the control cell), but these cells still contain focal adhesions and ventral stress fibers (examples are indicated by orange arrowheads in the VASP-depleted and control cell). However, the ventral stress fibers are abnormally aligned in VASP knockdown cells. The cells were stained with VASP antibody, vinculin antibody (to visualize focal adhesions) and phalloidin (to visualize F-actin). The VASP depleted cell is surrounded by a dashed line. Bar, 10 μm. (B) VASP depleted cells still exhibit ventral stress fibers, but they are predominantly poorly aligned and form abnormal clusters (orange arrowheads). Time-lapse frames from a movie of VASP-depleted U2OS cell expressing GFP-actin also demonstrate that in the absence of VASP (and visible dorsal stress fibers), ventral stress fibers (orange arrowheads) form from transverse arcs very close to the lamellipodium. Bar, 10 μm. (C) Over-expression of constitutively active (Ser239Ala;Thr278Ala) VASP affects elongation of stress fibers and often leads to a formation of abnormally curly actin filament bundles (visualized by phalloidin staining). Bar, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.06126.017