Figure 5. Elongation of dorsal stress fibers is regulated by VASP phosphorylation.
(A) U2OS cells treated with cAMPK and PKA inhibitors, compound C and KT5823, display abnormally long dorsal stress fibers (red arrows). Also fusion of arcs appears defective in compound C and KT5823 treated cells, because thick actin bundles (yellow arrowhead in the control cell) are largely absent from these cells. Actin filaments were visualized by phalloidin. Bar, 10 μm. (B) Quantification of dorsal stress fiber lengths (μm) from control cells and cells treated with compound C or KT5823. Mean lengths (+/- SEM) of 40 dorsal stress fibers from each sample are shown. (C) Western blot demonstrating that lysates of compound C or KT5823 -treated cells display decreased phosphorylation of VASP at Ser239 and Thr278. GADPH and total VASP were probed as loading controls. (D) Expression of constitutively active VASP mutant (Ser239Ala;Thr278Ala) induces formation of abnormally long dorsal stress fibers, whereas similar phenotype was not observed in wild-type VASP expressing cells. Bar, 10 μm. (E) Quantification of dorsal stress fiber lengths from cells expressing wild-type and Ser239Ala;Thr278Ala mutant VASP. Mean lengths (+/- SEM) of 55 dorsal stress fibers measured from both samples are shown.