Mechanical control of nuclear import by Importin-7 is regulated by its dominant cargo YAP

Mechanical forces regulate multiple essential pathways in the cell. The nuclear translocation of mechanoresponsive transcriptional regulators is an essential step for mechanotransduction. However, how mechanical forces regulate the nuclear import process is not understood. Here, we identify a highly mechanoresponsive nuclear transport receptor (NTR), Importin-7 (Imp7), that drives the nuclear import of YAP, a key regulator of mechanotransduction pathways. Unexpectedly, YAP governs the mechanoresponse of Imp7 by forming a YAP/Imp7 complex that responds to mechanical cues through the Hippo kinases MST1/2. Furthermore, YAP behaves as a dominant cargo of Imp7, restricting the Imp7 binding and the nuclear translocation of other Imp7 cargoes such as Smad3 and Erk2. Thus, the nuclear import process is an additional regulatory layer indirectly regulated by mechanical cues, which activate a preferential Imp7 cargo, YAP, which competes out other cargoes, resulting in signaling crosstalk.

List of the importins and exportins identified in the MS analysis (Fig. 1b), sorted according to the Z-score. From left to right, the columns show the proteins ID (Protein), the average nuclear amount in high confluence (high confl.) or low confluence (low confl.), the t-test p-value obtained from the comparison of high confl. nuclear and low confl. nuclear (T-test, in column 4), the average cytosolic amount in high confluence (high confl.) and low confluence (low confl.), the t-test p-value obtained from comparing high confl. cytosol and low confl. cytosol (T-test, column 7) and the Z-score value (Z; explained in figure legend 1b).
Statistical analysis with a two-tailed unpaired t test. Raw data is provided in Supplementary Data 1. a-j) In the a, c, e, g, i panels the immunoblot of the indicated proteins upon silencing with the indicated siRNAs is shown. GAPDH was used as loading control. In the adjacent panels to the immunoblots (b, d, f, h and j) the staining of the protein of interest is shown in control and cells treated with the corresponding siRNA. Representative of 3 independent experiments. Scale bar 10 µm. Quantification is shown in the right graph (d). N = 137 cells in soft condition and 123 cells in stiff matrix from 3 independent experiments. P-value = 1.958e-07.

c)
In situ PLA detection of the interaction between endogenous YAP and Imp7 in RPE-1 cells silenced with control, two independent siRNAs for Imp7 or two independent siRNAs for YAP. Cells were stained for anti-YAP (63.7) and anti-Imp7 (rabbit) antibodies. N = 9 fields from 3 independent experiments for each sample. From left to right, p-values = 7.534e-06, 1.539e-06, 6.259e-07 and 6.165e-07.

d)
Quantification of the levels of Imp7/GAPDH upon YAP silencing. N = 3 biologically independent experiments. Statistical analysis with a two-tailed unpaired t test. Data represent mean ± s.e.m. 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. Raw data available in the Source Data file.  * * Impb1 Impa1 Impa3 Impa4 Impa5 Impa6 Impa7 Impa8 Imp4 Imp5 Imp7 Imp8 Imp9 Imp11 Imp13 Xpo4 Tpno1 Tpno2 Tpno3 a) Quantification of the ratio between Imp7 protein levels and GAPDH levels (from Fig. 5a). The ratio was normalized to siControl. Data from 3 independent experiments. b) Quantification of the ratio between YAP protein levels and GAPDH levels (from Fig. 5a). The ratio was normalized to siControl. Data from 3 independent experiments. c) qRT-PCR analysis of YAP expression in RPE-1 cells silenced with control or two independent Imp7 siRNAs. Data were normalized to cells silenced with control siRNA. Data from 3 independent experiments. d) Quantification of pS127 YAP normalized to the total YAP protein levels upon silencing of Imp7 with two independent siRNAs.
The ratios were normalized to siControl. Data from 3 independent experiments. GAPDH was used as loading control. Statistical analysis with a two-tailed unpaired t test. Data represent mean ± sem.
Representative of 3 biologically independent experiments. Scale bar 10 µm. 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. Raw data available in the Source Data file. i, j) Immunofluorescence of endogenous Msk and nuclei in fully spread S2 cells plated for 24h in concanavalin A coated surfaces at low or high confluency. Quantification of each signal is shown in graph (j). N = 88 (high) and 78 (low) cells per condition, from 3 independent experiments. P-value = 2.51e-41. Scale bar 10 μm. k, l) S2 cells overexpressing different combinations of Msk-YFP, Yki-Flag(3x), YkiS168A-Flag(3x) and Yki ΔC-Flag(3x) were grown without substrate, therefore in semi-adherent conditions. Lysates were made and Msk-YFP was immunoprecipitated with an anti-GFP antibody. The immunopurified complexes and total cell lysates were immunoblotted with anti-Flag and anti-GFP antibodies as indicated. IgG was marked with an asterisk and Yki-Flag(3x) with a #. Quantification from three independent coimmunoprecipitations is shown in (l). P-value = 0.0039. Statistical analysis was performed with a two-tailed unpaired t test. Data represent mean ± s.e.m. 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. Raw data available in the Source Data file. Supplementary Fig. 7. Regions in YAP required to interact with Imp7 in cells and needed for nuclear accumulation. a, b, c) RPE-1 cells stably expressing the indicated proteins were lysed and endogenous Imp7 was immunoprecipitated. The immunopurified complexes and total cell lysates were immunoblotted with anti-GFP and anti-Imp7 antibodies as indicated. * indicates heavy and light IgG chains. # indicates the region where GFP runs, and it is not detected in the Imp7 immunoprecipitated fractions. In situ PLA detection of the association between endogenous Smad3 and Imp7 in RPE-1 cells silenced with control, two independent siRNAs for Imp7 or one siRNA for Smad3, using anti-Imp7 (mouse) and anti-Smad3 (rabbit) antibodies; quantification of the PLA signal is shown in panel b. For each sample, N = 9 fields from 3 independent experiments. From left to right, p-value = 7.35e-07, 1.27e-05 and 5.49e-07.

c, d)
In situ PLA detection of the association between endogenous Smad3 and Imp7 in RPE-1 cells treated with (+) or without (-) TGFβ, using anti-Imp7 (mouse) and anti-Smad3 (rabbit) antibodies; quantification of the PLA signal is shown in panel d. For each sample, N = 11 fields from 3 independent experiments. P-value = 1.70e-05. e, f) In situ PLA detection of the association between endogenous YAP and Imp7 in RPE-1 cells silenced with control or Smad3 siRNA, using anti-YAP (mouse) and anti-Imp7 (rabbit) antibodies. Quantification of the PLA signal is shown (f). For each sample, N = 9 fields from 3 independent experiments. P-value = 0.311.

m, n)
In situ PLA detection of the association between endogenous Erk2 and Imp7 in RPE-1 cells silenced with control or two independent siRNAs of YAP. Cells were treated with EGF (30 nM 30 min) and stained with anti-Imp7 (mouse) and anti-Erk2 (rabbit) antibodies. Quantification of the PLA signal is shown (n). For each sample, N = 5 fields from 3 independent experiments.
Statistical analysis with a two-tailed unpaired t test. Data represent mean ± s.e.m. From left to right, p-value = 0.032 and 0.028.
Scale bar 10 µm. 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. Raw data available in the Source Data file.