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. 2017 Jan 25;4(1):84–96.e6. doi: 10.1016/j.cels.2016.11.015

Figure 4.

Figure 4

YAP Is Activated by β-PIX through Focal-Adhesion-Dependent and Focal-Adhesion-Independent Pathways during Cell Spreading

(A) GFP-β-PIX in wild-type MCF10A cells. Scale bar, 20 μm.

(B) β-PIX may couple YAP activation to focal adhesions (i), regulate YAP independently of focal adhesions (ii), or activate YAP indirectly by promoting focal adhesion turnover (iii).

(C) Cells plated on fibronectin (FN) in the presence of DMSO (control) or small-molecule inhibitors of FAK (PF573288), ROCK (H1152), and myosin II (blebbistatin). Green, YAP; purple, F-actin. Scale bar, 20 μm.

(D) Focal adhesion area (normalized to cell area) in cells plated on FN for 4 hr. Mean ± 95% CI (n = 30–80 cells/condition).

(E) Nuclear/cytoplasmic YAP ratio as a function of cell area in cells plated on FN (4 hr) in the presence of DMSO or FAKi (PF-573288). Mean ± SD of replicate wells (n = 4 wells/condition, 230 ± 95 cells/well).

(F) Inhibiting FAK kinase activity blocks turnover of focal contacts and promotes maturation and growth of focal adhesions. FAK-independent YAP activation requires β-PIX, Cdc42, Rac1, and PAK2.

(G and H) Nuclear/cytoplasmic YAP ratio as a function of cell area in cells plated on FN (4 hr) in the presence of DMSO (open shapes) or inhibitors of ROCK (G) or myosin II (H) (filled shapes). Mean ± SD of replicate wells (n = 4 wells/condition, 230 ± 95 cells/well).

(I) Inhibiting ROCK kinase or myosin II ATPase activity blocks focal adhesion maturation and promotes contact formation. Myosin-II-independent YAP activation requires β-PIX and Cdc42.