The mechanical stability of proteins regulates their translocation rate into the cell nucleus

Abstract

The translocation of mechanosensitive transcription factors (TFs) across the nuclear envelope is a crucial step in cellular mechanotransduction. Yet the molecular mechanisms by which external mechanical cues control the nuclear shuttling dynamics of TFs through the nuclear pore complex (NPC) to activate gene expression are poorly understood. Here, we show that the nuclear import rate of myocardin-related transcription factor A (MRTFA) - a protein that regulates cytoskeletal dynamics via the activation of the TF serum response factor (SRF) - inversely correlates with the protein's nanomechanical stability and does not relate to its thermodynamic stability. Tagging MRTFA with mechanically resistant proteins results in the downregulation of SRF-mediated myosin light-chain 9 (MYL9) gene expression and subsequent slowing down of cell migration. We conclude that the mechanical unfolding of proteins regulates their nuclear translocation rate through the NPC, and highlight the role of the NPC as a selective mechanosensor able to discriminate forces as low as ~10 pN. The modulation of the mechanical stability of TFs may represent a new strategy for the control of gene expression.

Figure. 1. The kinetics of MRTFA nuclear import is regulated by its mechanical properties.

(a) Representative confocal image gallery showing MRTFA nuclear translocation, upon serum stimulation, in a U2OS cell stably expressing MRTFA-GFP. 10μm scale bar. (b) Individual (light grey lines) and averaged (green points, mean ± s.e.m.) time courses of nucleus/cell MRTFA. These translocation dynamics can be fit to n(t) = n_e(1 – e^–kt) (black line), yielding an equilibrium accumulation n_e = 0.784 ± 0.019 and a total rate constant k = 3.91 ± 0.42 ×10^-3 s^-1, which can be decomposed into import and export rate constants k_I = 3.07 ± 0.32 ×10^-3 s^-1 and k_E = 0.84 ± 0.14 ×10^-3 s^-1, respectively. Data are from four independent experiments (n = 24). (c) Structural schematic of titin, showing the distribution of selected Ig domains along the N-C termini direction. (d) Schematics of a single molecule force spectroscopy experiment, whereby a polyprotein made of selected titin Ig domains is tethered between an AFM cantilever tip and a gold substrate. (e) Stretching individual (Ig1-Ig27_C47A-C63A)₄, (Ig27)₈ (not shown here), and (Ig32)₈ polyproteins at a constant velocity of 400 nm s^-1 gives rise to unfolding trajectories exhibiting saw-tooth patterns, where each force peak corresponds to the unfolding of an individual Ig domain within the polyprotein chain. (f) Probability density histograms of unfolding forces for Ig1 (yellow), Ig27 (grey), and Ig32 (magenta) domains with the associated Gaussian probability density distributions (black) overlaid (unfolding force mean ± s.d.: Ig1, 144 ± 27 pN, n = 137; Ig27, 208 ± 28 pN, n = 186; Ig32, 267 ± 33 pN, n = 936). (g) Inserting Ig domains within MRTFA constructs enables one to probe the effect of mechanical stability on nuclear translocation. (h) Averaged (mean ± s.e.m.) time courses of nucleus/cell MRTFA in U2OS cells expressing MRTFA-Ig1-BFP, MRTFA-Ig27-BFP, or MRTFA-Ig32-BFP, after serum stimulation. The rate and extent of MRTFA nuclear translocation of the mechanically-labile Ig1 is higher than that of Ig27 and of the mechanically-stable Ig32. Data are from four independent experiments (Ig1, n = 34; Ig27, n = 37, Ig32, n = 44), only a representative error bar is shown per condition. (i) Total (squares), import (right-pointing triangles), and export (left-pointing triangles) rate constants associated with the nuclear translocation of Ig-domain-tagged MRTFA constructs plot against the mechanical stability (unfolding force) of the tagging Ig domain. The import rate constant displays exponential dependence (R² = 0.999) with mechanical stability. By contrast, the export rate constant is largely independent of mechanical stability. Rate constants correspond to the fitting parameters from the averaged time courses in (h), associated error bars correspond to the s.e.m. of fitting parameters of the individual time courses. The unfolding forces correspond to the mean ± s.d. of unfolding forces as in (f). Dashed lines are a weighted linear fit (import) and average (export).

Figure. 2. The nuclear pore complex is highly mechanoselective.

(a) Selective introduction of point mutations in key residues in the mechanical-clamp region of the Ig27 structure result in subtle changes to its mechanical stability, in the order V13P < V11P < V15P < WT < Y9P, as measured elsewhere by single molecule force spectroscopy experiments. (b) Averaged (mean ± s.e.m.) time courses of MRTFA nuclear translocation, upon serum stimulation, in U2OS cells expressing MRTFA-GFP tagged with different Ig27 mutants – the rate and extent of MRTFA nuclear accumulation is dependent on the tagging Ig27 variant. Data are from two (V11P, n = 26) or three (V13P, n = 22; V15P, n = 25; WT, n = 30; Y9P, n = 17) independent experiments, only a representative error bar is shown per condition. (c) The import rate constant (triangles) displays an exponential dependence (dashed line, linear fit, R² = 0.988) with the mechanical stability of the tagging Ig27 mutant. By contrast, a weaker correlation is observed with their thermodynamic stability (circles). Import rate constants correspond to the fitting parameters from averaged time courses, associated error bars correspond to the s.e.m. of fitting parameters from individual time courses. Melting temperatures correspond to the mean of 12 replicates (with associated s.e.m. < 0.1 °C for each mutant, not shown). Unfolding forces correspond to the mean ± estimated s.d. (20 pN) as in (a). (d) Averaged melting curves of Ig27 variants (colour coded as in (a)) from differential scanning fluorimetry experiments. (e) Representative confocal image galleries showing MRTFA nuclear translocation, following serum stimulation, of a U2OS cell co-expressing MRTFA-I27_WT-BFP and MRTFA-I27_V11P-YFP. 10 μm scale bar. (f) Corresponding averaged (mean ± s.e.m.) time courses showing that, within the same cell, the rate and extent of nuclear accumulation of MRTFA tagged with the mechanically-labile Ig27_V11P is higher than MRTFA tagged with the mechanically-stable Ig27_WT. Data are from three independent experiments (n = 9), only a representative error bar is shown per condition. (g) Ratios (mean ± s.e.m.) of import (right-pointing triangles) and export (left-pointing triangles) rate constants plot against the unfolding force differences for co-expression experiments (full details in Fig. S10). Data are from three independent experiments (left to right, BFP/YFP: V13P/V13P, n = 9; V15P/V13P, n = 8; WT/V11P, n = 9; WT/V13P, n = 15; Y9P/V13P, n = 12). Ratios correspond to the mean ± s.e.m. of the ratios of the fitting parameters associated with translocation of the BFP construct, with respect to the YFP construct, from each individual cell. Dashed lines are a weighted linear fit (import) and average (export).

Figure. 3. Physical deformation of the nucleus is a complementary pathway to induce MRTFA nuclear translocation.

(a) Representative epifluorescence images of U2OS cells stably expressing MRTFA-GFP, plated on 6 or 81 kPa polyacrylamide gels or glass. 10 μm scale bars. (b) The nuclear accumulation of MRTFA-GFP increases significantly with substrate stiffness, a trend that is abolished when treating cells with latrunculin B. Data are from three independent experiments (DMSO: 6 kPa, n = 167; 81 kPa, n = 166; glass n = 162. Lat B: 6 kPa, n = 164; 81 kPa, n = 145; glass, n = 159). Two-way ANOVA, NS P > 0.05, ** P ≤ 0.01, and **** P ≤ 0.0001. (c) Analysis of nuclear heights via measuring the full-width half-maximum of fluorescent intensity on vertical sections through XZ sum projections of DAPI-stained nuclei. The inset shows representative XZ projections (top, 6 kPa; bottom, 81 kPa). Nuclear heights are significantly increased on 6 kPa gels compared to 81 kPa gels, concluding that the nuclear envelope is more deformed on stiffer substrates. 5 μm scale bar. Data are from two independent experiments (6 kPa, n = 49; 81 kPa, n = 26). Two-tailed t-test, **** P≤ 0.0001. (d) Representative epifluorescence image gallery of a U2OS cell stably expressing MRTFA-GFP, showing MRTFA nuclear translocation triggered by the application of 1 nN by a pyramidal AFM tip. (e) MRTFA nuclear translocation occurs in a force-dependent manner. Data points represent the mean ± s.e.m. of multiple experiments (0.5 nN, n = 7; 1 nN, n = 15; 5 nN, n = 10). (f) Localized nuclear deformation is induced by the AFM tip, as confirmed by vertical image slices of NLS-mCherry, dashed lines represent an estimation of the (diagonal) tip profile. (g) Averaged (mean ± s.e.m.) time courses of MRTFA nuclear translocation in U2OS cells, following serum stimulation, comparing the dynamics of MRTFA-Ig27_WT-BFP and MRTFA-Ig27_V11P-YFP when cells are plated on glass or 6 kPa gel substrates, revealing that, although nuclear accumulation is significantly decreased on 6 kPa gels (mostly due to a 4-fold increase in the export rate constant (Fig. S11)), the mechanical selectivity to the translocating protein remains unaltered. Data are from three independent experiments (glass, n = 15; 6 kPa gel, n = 13), only a representative error bar is shown per condition.

Figure. 4. Mechanically-stable MRTFA constructs downregulate gene expression and cellular motility.

(a) Real-time quantitative PCR in U2OS cells stably expressing a GFP empty vector, MRTFA-GFP, MRTFA-Ig27_WT-GFP or MRTFA-Ig27_V13P-GFP, 4 hours after serum stimulation. MYL9 gene expression is significantly increased when MRTFA is tagged with the mechanically-labile Ig27_V13P domain compared to the mechanically-stable I27_WT. Data are from three independent experiments, bars show mean ± s.e.m. (b) Wound-healing assays on U2OS stable cell lines show that wound healing is significantly slower in cells expressing MRTFA-Ig27_WT-GFP compared to MRTFA-Ig27_V13P-GFP. Data are from three independent experiments (GFP empty vector, n = 21; MRTFA-GFP, n = 23; MRTFA-Ig27_V13P-GFP, n = 24; MRTFA-Ig27_WT-GFP, n = 24), bars show mean ± s.e.m. (c) Representative bright-field images of wound healing of MRTFA-Ig27_V13P-GFP and MRTFA-Ig27_WT-GFP stable cell lines. 100 μm scale bar. (d) Motility assays of MDA-MB-231 cells showing that the migration speed of cells transfected with MRTFA-Ig27_V13P-GFP is significantly higher than those transfected with MRTFA-Ig27_WT-GFP. Data are from three independent experiments (GFP empty vector, n = 101; MRTFA-GFP, n = 74; MRTFA-Ig27_V13P-GFP, n = 92; MRTFA-Ig27_WT-GFP, n = 98), bars show mean ± s.e.m. All statistical tests are two-tailed t-tests, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. (e) Schematic representation of the proposed nuclear import mechanism by which the mechanical properties of the translocating protein regulate their nuclear shuttling dynamics.