Fig. 10: YAP restricts Smad3 and Imp7 association.
From: Mechanical control of nuclear import by Importin-7 is regulated by its dominant cargo YAP
a–d In situ PLA detection of the association between endogenous Smad3 and Imp7 in RPE-1 cells growing at low (a, b) or high confluence (c, d) and silenced with control or two independent siRNAs for YAP. Quantification of the PLA signal is in panels (b) and (d). N = 9 fields (low confluence) and N = 12 fields (high confluence) from 3 independent experiments. In panel b: P-value = 0.0011 (1st vs. 2nd lanes) and 0.00079 (1st vs. 3rd lanes). In panel d: p-value = 0.311 (1st vs. 2nd lanes), 0.076 (1st vs. 3rd lanes), and 0.563 (2nd vs. 3rd lanes). e, f In situ PLA detection of the association between endogenous Smad3 and Imp7 in RPE-1 cells overexpressing the indicated proteins and growing at the low confluence. f Quantification of the PLA signal. N = 16 fields in each sample from 3 independent experiments. P-value = 8.563e−10 (1st vs. 2nd bars), 0.541 (1st vs. 3rd), 0.336 (1st vs. 4th), 1.437e−11 (2nd vs. 4th), and 0.726 (3rd vs. 4th). g Immunoblot showing the levels of overexpressed EYFP-YAP, EYFP-YAP S127E, and EYFP-YAP ΔC mutants in RPE-1 cells. h, i In situ PLA detection of the association between endogenous Smad3 and Imp7 in RPE-1 cells growing at the high confluence and treated with (+) or without (−) XMU-MP-1 for 16 h. i Quantification of the PLA signal. N = 13 fields for each sample from 3 independent experiments. Statistical analysis with a two-tailed unpaired t-test. Data represent mean ± s.e.m. Scale bar 10 µm. P-value = 2.014e−07. Raw data are available in the Source Data file. P-values below or equal to 0.05, 0.01, or 0.005 were considered statistically significant and were labeled with 1, 2, or 3 asterisks, respectively. j A graphical model to explain the results of this study. In conditions of low cell tension, such as high cell density or disruption of the actin cytoskeleton, YAP is no longer competent to bind Imp7 due to phosphorylation on YAP serine 127. This setting favors Smad3 and Erk2 association with Imp7, which results in more nuclear Smad3 and Erk2. On the contrary, under conditions of high cell tension, such as low cell density and intact contractile actin cytoskeleton, the Hippo pathway is inactive, resulting in the formation of the Imp7–YAP complex, which drives YAP and YAP-dependent Imp7 nuclear localization. Under these conditions, the association of Smad3 and Erk2 to Imp7 is limited by YAP. This mechanism permits the regulation of other signaling pathways by the Hippo pathway in a cell tension-dependent manner by the competition of YAP for Smad3 or Erk2 access to Imp7. Created using Biorender.com.
