Figure 2.

YAP is regulated by the actin and microtubule cytoskeletons. (A) Cell attachment-induced YAP dephosphorylation requires actin polymerization. MCF10A cells were trypsinized (T) and attached onto fibronectin-coated petri dishes (A) for 80 min. Latrunculin B was added as indicated at the time of plating. Cell lysates were analyzed by Western blots as indicated. (B) Microtubule organization is required for cell detachment-induced YAP phosphorylation. MCF10A cells were trypsinized and attached onto fibronectin-coated petri dishes for 2 h. Cells were either directly lysed (A) or trypsinized again before being collected and lysed (T). Nocodazole (Noco) and taxol were added to cell culture medium 15 min before and during trypsinization as indicated. (C) Disruption of the microtubule decreases YAP phosphorylation. MCF10A cells cultured to 80% confluence were treated with the indicated chemicals in culture medium. Cells were then lysed and analyzed by Western blots. (D) Disruption of the actin cytoskeleton induces YAP phosphorylation. MCF10A cells were trypsinized and attached onto fibronectin-coated petri dishes for 2 h, followed by addition of the indicated reagents, and were further cultured for another hour before being harvested for Western blot analysis. (E) F-actin is required for YAP dephosphorylation induced by microtubule depolymerization. MCF10A cells were cultured to confluence and treated with inhibitors in cultured medium as indicated. Cell lysates were resolved on Phos-tag-containing SDS-PAGE gels, and anti-YAP antibody was used for Western blotting. Data were cropped from the same exposure of the same blot. (F) The actin and microtubule cytoskeletons regulate YAP subcellular localization. MCF10A cells were cultured onto fibronectin-coated coverglasses at low or high cell density. Cells were treated with the indicated inhibitors before fixation. Samples were then stained with anti-YAP antibody for endogenous YAP, Alexa Fluor 488-conjugated phalloidin for F-actin, and DAPI for cell nuclei. (G) Rho family small GTPases induce YAP dephosphorylation. HEK293 cells were cotransfected with the indicated plasmids. Cells were then lysed and analyzed by Western blots. (H) Rho mediates cell attachment-induced YAP dephosphorylation. MCF10A cells were trypsinized and then attached to fibronectin- or polylysine-coated petri dishes for 2 h in the presence of Rho inhibitor C3 (2 μg/mL) or Rac inhibitor NSC23766 (100 μM). Cell lysates were resolved on Phos-tag-containing SDS-PAGE gels, and anti-YAP antibody was used for Western blotting.