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. Author manuscript; available in PMC: 2023 Jul 21.
Published in final edited form as: Nat Phys. 2019 Jul 1;15(9):973–981. doi: 10.1038/s41567-019-0551-3

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

Figure. 1

(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) = ne(1 – e–kt) (black line), yielding an equilibrium accumulation ne = 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 kI = 3.07 ± 0.32 ×10-3 s-1 and kE = 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-Ig27C47A-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).