Figure 4. YAP interacted with β-catenin to bias stiffness-mediated NSC lineage commitment.

A: YAP-GFP decreased β-catenin transcriptional activity. The activity of a β-catenin responsive promoter-reporter was significantly reduced in YAP-GFP cells compared to control cells (n = 3, p<0.01 by t-test). B, C: In control cells (B), YAP-GFP cells did not stain positive for βIII-tubulin, but GSK3β inhibition restored neural differentiation in some YAP-GFP cells (C). Yellow stars indicate cells that stain positive for both YAP-GFP and βIII-tubulin. D: YAP and β-catenin interact in differentiating NSCs. Co-immunoprecipitation with a β-catenin antibody and probing with a YAP antibody showed that on both 300 Pa (soft) and 72 kPa (stiff) samples, YAP co-precipitated with β-catenin (control: no β-catenin antibody on stiff surface). E,F: Binding mutants of YAP-GFP showed differentiation rescue of neurogenesis on soft (200 Pa) substrates. A YAP mutant unable to bind β-catenin (E66A) (E) showed the same level of neuronal differentiation as naïve, non-YAP expressing cells, compared to suppressed neurogenesis by YAP-GFP (compare with B). Numerous cells stained positive for both βIII-tubulin and E66A YAP-GFP; in contrast, in the control YAP-GFP case there were no cells that stained positive for both βIII-tubulin and GFP. The S94A YAP mutant that lacked TEAD binding (F) suppressed neurogenesis to the same extent as wild type YAP-GFP (compare B). G: Quantification of levels of neurogenesis in the naïve, E66A, S94A, and YAP-GFP cases. Naïve and E66A were significantly different from S94A and YAP-GFP cases on soft substrates (200 Pa) (n=3 gels, p < 0.005 by 1-way ANOVA). H: Schematic of the proposed effect of substrate stiffness on NSC lineage commitment. On soft substrates, β-catenin drives transcription of neurogenesis effectors, such as NeuroD1. On stiff substrates, YAP levels are sufficiently high to sequester and inhibit available β-catenin, thereby preventing β-catenin dependent transcription.