Mechanomemory after short episodes of intermittent stresses induces YAP translocation via increasing F-actin

Abstract

How forces and mechanics influence and regulate living cells remains elusive. Mechanomemory, the response to a mechanical perturbation that persists after the perturbation is removed, is believed to be a key to understanding the impact of forces and mechanics on cell functions. Recently, our lab has demonstrated the presence of mechanomemory that lasts for ∼30 min after applying external stress via integrins. Herein, we test the hypothesis that applications of short intermittent episodes of stress exert long-term effects on mechanomemory via the process of mechanotransduction. An Arginine-Glycine-Aspartic acid (RGD)-peptides-coated 4-μm magnetic bead was bound to the integrin receptors to apply stresses to the surface of a Chinese Hamster Ovary cell. At the same stress magnitude and frequency (15 Pa at 0.3 Hz), multiple cycles of externally applied intermittent 2 or 10 min stresses with 15 min intervals, 10 min stresses with 10 min intervals, or a 30 min stress plus a 30 min load-free interval increased nuclear translocation of YAP (Yes-Associated Protein) and Ctgf gene expression, like that by a 60 min continuous stress, but a 30 min continuous stress did not. Short durations of intermittent stresses increased F-actin in the cytoplasm, which coincided with the elevated YAP translocation. Inhibiting F-actin or actomyosin but not microtubules blocked stress-induced YAP translocation to the nucleus. Cells on soft substrates translocate more YAP than on stiff substrates after external load release. These results highlight the impact of multiple intermittent stresses-induced cytoplasmic mechanomemory on cell biological functions via YAP translocation.

FIG. 1.

Multiple short intermittent stresses induce YAP/TAZ nuclear translocation via cytoplasmic mechanomemory. (a) Schematic of a 4-μm Arginine-Glycine-Aspartic acid (RGD)-coated ferromagnetic bead binds to the integrin receptors of Chinese Hamster Ovary (CHO) cell to apply stress by magnetizing in Y-direction and applied twisting field (all stresses were applied with 15 Pa at 0.3 Hz) in Z-direction. (b) Multiple sinusoidal stresses of 2 or 10 min with 15 min or 10 min intervals, continuous sinusoidal stresses of 30 or 60 min, and a 30 min interval after 30 min continuous stress to quantify YAP/TAZ nuclear translocation and mechanomemory. (c) Representative brightfield (BF) and YAP/TAZ immunofluorescence (IF; red) images (Anti-YAP/TAZ primary antibody and Anti-Rabbit IgG H&L (Alexa Fluor^® 555) secondary antibody) of CHO cells at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60 min, and 30 min interval after 30 min continuous (30 + 30 min) stress. The black dot was an RGD-coated ferromagnetic bead. Scale bar, 10 μm. (d) Normalized YAP/TAZ nuclear to cytoplasmic (nuclear/cytoplasmic) intensity ratio for 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 55 cells for 0 min, n = 45 cells for 2 + 15 min, n = 48 cells for 10 + 10 min, n = 45 cells for 10 + 15 min, n = 61 cells for 30 min, n = 56 cells for 30 + 30 min, and n = 42 cells for 60 min stress condition from three independent experiments; P = 0.019 between 0 and 2 + 15 min; P = 2 × 10⁻⁴ between 0 and 10 + 10 min; P = 1.74 × 10⁻⁵ between 0 and 10 + 15 min; P = 0.022 between 2 + 15 and 10 + 15 min; P = 2.30 × 10⁻⁷ between 0 and 60 min; P = 0.0013 between 0 and 30 + 30 min; P = 0.018 between 30 and 30 + 30 min; P = 0.0034 between 30 + 30 and 60 min; P = 0.008 between 10 + 10 and 60 min. (e) qPCR analysis of Ctgf expression for 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress. Data are shown as bar plot with mean ± s.e.m. values from three independent biological experiments. P = 0.014 between 0 and 2 + 15 min; P = 0.019 between 0 and 10 + 10 min; P = 0.005 between 0 and 10 + 15 min; P = 0.01 between 0 and 60 min; P = 0.013 between 0 and 30 + 30 min; P = 0.027 between 2 + 15 and 10 + 15 min; P = 0.033 between 30 and 30 + 30 min; P = 0.021 between 30 and 60 min. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns, not significantly different. All P values were quantified using one-way ANOVA with Tukey's test and Mann–Whitney test.

FIG. 2.

Multiple short intermittent stresses increase F-actin and disrupting F-actin abolishes YAP/TAZ translocation. (a) Representative brightfield (BF) and F-actin stained (SiR-actin) fluorescence images (red) of live CHO cells at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60, and 30 min interval after 30 min continuous stress (30 + 30 min); all stresses were 15 Pa at 0.3 Hz. Circular black dots are RGD-coated ferromagnetic beads. Scale bar, 10 μm. (b) Normalized F-actin fluorescence intensity for 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress of 15 Pa at 0.3 Hz. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 105 cells for 0 min, n = 87 cells for 2 + 15 min, n = 57 cells for 10 + 10 min, n = 77 cells for 10 + 15 min, n = 115 cells for 30 min, n = 64 cells for 30 + 30 min, and n = 99 cells for 60 min stress condition from three independent experiments; P = 0.032 between 0 and 2 + 15 min; P = 0.0001 between 0 and 10 + 10 min; P = 2.46 × 10⁻⁸ between 0 and 10 + 15 min; P = 5.34 × 10⁻⁵ between 2 + 15 and 10 + 15 min; P = 1.50 × 10⁻⁸ between 0 and 60 min; P = 0.014 between 2 + 15 and 30 min; P = 0.0011 between 2 + 15 and 60 min; P = 4.50 × 10⁻⁸ between 10 + 15 and 30 min; P = 3.26 × 10⁻⁸ between 30 and 60 min; P = 8.22 × 10⁻⁸ between 0 and 30 + 30 min; P = 0.0011 between 2 + 15 and 30 + 30 min; P = 2.01 × 10⁻⁸ between 30 and 30 + 30 min; P = 0.0032 between 10 + 10 and 10 + 15 min. (c) Representative brightfield (BF) and YAP/TAZ immunofluorescence (IF; red) images (used YAP/TAZ antibody and Anti-Rabbit IgG H&L (Alexa Fluor® 555) antibody) of CHO cells treated with 1 μM Latrunculin A (Lat A) for 30 min at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60, and 30 min interval after 30 min continuous (30 + 30 min) stress of 15 Pa at 0.3 Hz. Circular black dots are RGD-coated ferromagnetic bead. Scale bar, 10 μm. (d) Normalized YAP/TAZ nuclear to cytoplasmic (nuclear/cytoplasmic) intensity ratio in 1 μM Latrunculin A (Lat A)-treated CHO cells for 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 102 cells for 0 min, n = 111 cells for 2 + 15 min, n = 46 cells for 10 + 10 min, n = 76 cells for 10 + 15 min, n = 115 cells for 30 min, n = 63 cells for 30 + 30 min, and n = 65 cells for 60 min stress condition from three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns, not significantly different. All P values were quantified using one-way ANOVA with Tukey's test and Mann–Whitney test.

FIG. 3.

Inhibiting non-muscle myosin II block mechanomemory-mediated YAP/TAZ nuclear translocation. (a) Representative brightfield (BF) and YAP/TAZ immunofluorescence (IF; red) images (used YAP/TAZ antibody and Anti-Rabbit IgG H&L (Alexa Fluor® 555) antibody) of CHO cells treated with 20 μM Blebbistatin for 30 min at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60, and 30 min interval after 30 min continuous (30 + 30 min) stress of 15 Pa at 0.3 Hz. Circular black dots are RGD-coated ferromagnetic beads. Scale bar, 10 μm. (b) Normalized YAP/TAZ nuclear to cytoplasmic (Nuclear/Cytoplasmic) intensity ratio of 20 μM Blebbistatin-treated CHO cells at 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress of 15 Pa at 0.3 Hz. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 60 cells for 0 min, n = 60 cells for 2 + 15 min, n = 48 cells for 10 + 10 min, n = 60 cells for 10 + 15 min, n = 85 cells for 30 min, n = 57 cells for 30 + 30 min, and n = 59 cells for 60 min stress condition from three independent experiments; P = 0.004 between 0 and 60 min; P = 1.59 × 10⁻⁴ between 2 + 15 and 60 min; P = 0.009 between 10 + 10 and 60 min; P = 0.023 between 10 + 15 and 60 min; P = 0.008 between 30 and 60 min; P = 1.19 × 10⁻⁴ between 30 + 30 and 60 min. (c) Quantified cell nuclear area of 20 μM Blebbistatin-treated CHO cells at 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress of 15 Pa at 0.3 Hz. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 60 cells for 0 min, n = 60 cells for 2 + 15 min, n = 48 cells for 10 + 10 min, n = 60 cells for 10 + 15 min, n = 85 cells for 30 min, n = 57 cells for 30 + 30 min, and n = 59 cells for 60 min stress condition from three independent experiments; P = 0.0014 between 0 and 60 min; P = 0.025 between 10 + 15 and 60 min.^*P < 0.05, ^**P < 0.01; ns, not significantly different. All P values were quantified using one-way ANOVA with Tukey's test and Mann–Whitney test.

FIG. 4.

Inhibiting microtubule does not block YAP/TAZ nuclear translocation by disrupting F-actin. (a) Representative brightfield (BF) and YAP/TAZ immunofluorescence (IF; red) images (used YAP/TAZ antibody and Anti-Rabbit IgG H&L (Alexa Fluor^® 555) antibody) of CHO cells treated with 10 μM Colchicine for 30 min at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60, and 30 min interval after 30 min continuous (30 + 30 min) stress of 15 Pa at 0.3 Hz. Circular black dots are RGD-coated ferromagnetic beads. Scale bar, 10 μm. (b) Normalized YAP/TAZ nuclear to cytoplasmic (nuclear/cytoplasmic) intensity ratio after 10 μM Colchicine-treated CHO cells exposed to 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress of 15 Pa at 0.3 Hz. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 79 cells for 0 min, n = 47 cells for 2 + 15 min, n = 49 cells for 10 + 10 min, n = 57 cells for 10 + 15 min, n = 49 cells for 30 min, n = 54 cells for 30 + 30 min, and n = 45 cells for 60 min stress condition from three independent experiments; P = 0.042 between 0 and 2 + 15 min; P = 2.77 × 10⁻⁵ between 0 and 10 + 10 min; P = 6.43 × 10⁻⁶ between 0 and 10 + 15 min; P = 0.033 between 2 + 15 and 10 + 15 min; P = 2.46 × 10⁻⁸ between 0 and 60 min; P = 0.03 between 0 and 30 + 30 min; P = 5.53 × 10⁻⁴ between 30 + 30 and 60 min. (c) Representative brightfield (BF) and F-actin stained (SiR-actin) fluorescence images (red) of live CHO cells at 0 min (no stress; control) before (left two images) and 30 min after (right two images) 10 μM Colchicine treatment. Cells were tracked before and 30 min after 10 μM Colchicine treatment. (d) Normalized F-actin fluorescence intensity of live CHO cells at 0 min (no stress; control) before and 30 min after 10 μM Colchicine treatment. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 48 cells for before and 30 min after 10 μM Colchicine treatment from three independent experiments. Inset shows F-actin intensity of each individual cell before and 30 min after 10 μM Colchicine treatment. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns, not significantly different. All P values were quantified using one-way ANOVA with Tukey's test and Mann–Whitney test.

FIG. 5.

Mechanomemory of YAP/TAZ nuclear translocation on soft and stiff substrates. (a) Representative brightfield (BF) and YAP/TAZ immunofluorescence (IF; red) images (used YAP/TAZ antibody and Anti-Rabbit IgG H&L (Alexa Fluor® 555) antibody) of CHO cells grown on a 1 kPa PA-gel at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60, and 30 min interval after 30 min continuous (30 + 30 min) stress of 15 Pa at 0.3 Hz. Circular black dots are RGD-coated ferromagnetic bead. Scale bar, 10 μm. (b) Normalized YAP/TAZ nuclear to cytoplasmic (Nuclear/Cytoplasmic) intensity ratio for 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress of 15 Pa at 0.3 Hz on 1 kPa PA-gel substrate. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 49 cells for 0 min, n = 34 cells for 2 + 15 min, n = 29 cells for 10 + 10 min, n = 52 cells for 10 + 15 min, n = 32 cells for 30 min, n = 35 cells for 30 + 30 min, and n = 34 cells for 60 min stress condition on 1 kPa PA-gel substrate from three independent experiments; P = 6.40 × 10⁻⁸ between 0 and 2 + 15 min; P = 4.16 × 10⁻⁸ between 0 and 10 + 10 min; P = 1.20 × 10⁻⁸ between 0 and 10 + 15 min; P = 0.0004 between 2 + 15 and 10 + 15 min; P = 6.48 × 10⁻⁸ between 0 and 60 min; P = 5.00 × 10⁻⁵ between 0 and 30 + 30 min; P = 0.04 between 30 and 30 + 30 min; P = 2.42 × 10⁻⁸ between 30 + 30 and 60 min; P = 0.006 between 10 + 10 min and 10 + 15 min. (c) Representative brightfield (BF) and YAP/TAZ immunofluorescence (IF; red) images (used YAP/TAZ antibody and Anti-Rabbit IgG H&L (Alexa Fluor® 555) antibody) of CHO cells grown on a 20 kPa PA-gel at 0 min (no stress; control), multiple 2 min (2 + 15 min) or 10 min with 10 min (10 + 10 min) or 15 min (10 + 15 min) interval, continuous 30 min or 60, and 30 min interval after 30 min continuous (30 + 30 min) stress of 15 Pa at 0.3 Hz. Circular black dots are RGD-coated ferromagnetic bead. Scale bar, 10 μm. (d) Normalized YAP/TAZ nuclear to cytoplasmic (nuclear/cytoplasmic) intensity ratio for 0, 2 + 15, 10 + 10, 10 + 15, 30, 30 + 30, or 60 min applied stress of 15 Pa at 0.3 Hz on 20 kPa PA-gel substrate. Data are shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean. n = 43 cells for 0 min, n = 31 cells for 2 + 15 min, n = 32 cells for 10 + 10 min, n = 37 cells for 10 + 15 min, n = 34 cells for 30 min, n = 34 cells for 30 + 30 min, and n = 34 cells for 60 min stress condition on 20 kPa PA-gel substrate from three independent experiments; P = 1.21 × 10⁻⁷ between 0 and 2 + 15 min; P = 4.05 × 10⁻⁸ between 0 and 10 + 10 min; P = 4.24 × 10⁻⁸ between 0 and 10 + 15 min; P = 0.008 between 2 + 15 and 10 + 15 min; P = 4.13 × 10⁻⁸ between 0 and 60 min; P = 7.33 × 10⁻⁵ between 0 and 30 + 30 min; P = 0.002 between 30 and 30 + 30 min; P = 0.000 03 between 30 + 30 and 60 min.^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns, not significantly different. All P values were quantified using one-way ANOVA with Tukey's test and Mann–Whitney test.