Fig. 6: Mechanically labile peptide tags accelerate the nuclear import of the mechanosensitive myocardin-related transcription factor (MRTFA), resulting in increased gene expression and motility of U2OS cells.
From: Structural anisotropy results in mechano-directional transport of proteins across nuclear pores
a, Representative confocal microscopy image gallery of U2OS doubly transfected with MRTFA-GFP–MRTFA-mCherry (left) and (GS)4-MRTFA-GFP–MRTFA-mCherry (right) at different time points after serum stimulation. Scale bars, 10 µm. b, Normalized average time courses of the relative nucleus-to-cytoplasm localization of MRTFA and (GS)4-MRTFA. The average time course is normalized by the average nucleus-to-cytoplasm accumulation (Ke) of the co-translocating MRTFA-mCherry construct. c, Associated normalized nuclear import rates: MRTFA (\({\widetilde{k}}_{\rm{I}}\) = 0.68 ± 0.03, n = 21); (GS)4-MRTFA (\({\widetilde{k}}_{\rm{I}}\) = 0.87 ± 0.03, n = 34), where \({\widetilde{k}}_{\rm{I}}\) is the normalized import rate calculated as kI(GFP)/kI(mCherry). Significance levels for the two-tailed Mann–Whitney non-parametric test: ****P < 0.0001; MRTFA versus (GS)4-MRTFA, P = 3.33 × 10−5. d, RT–qPCR in U2OS cells stably expressing the MRTFA-GFP and (GS)4-MRTFA-GFP, 4 h after serum stimulation (N = 7 independent experiments). Bars show mean ± s.d. Significance levels for the two-tailed paired t-test after log transformation: *P < 0.01; **P < 0.001; SRF, P = 7.16 × 10−3, MYL9, P = 0.04. e,f, U2OS cells stably expressing GS-tagged MRTFA exhibit increased cell motility: representative wound-healing assays on U2OS stable cell lines expressing MRTFA or (GS)4-MRTFA (scale bar, 100 µm) (e); relative wound-healing recovery calculated from the velocity of the cell front during the first 10 h for MRTFA (n = 74) and (GS)4-MRTFA (n = 62) (f). Significance levels for the two-tailed t-test: *P < 0.05 (P = 0.03). g, Free-energy surface for active nucleoplasmic transport projected against the molecular weight and the unfolding force (characterized by AFM) of the translocating protein. The free-energy difference was calculated as \({\Delta G(M,\,{F}_{\rm{U}})}={-{kT}\,\mathrm{ln}\frac{{k}_{\rm{I}}(M,\,{F}_{\rm{U}})}{{k}_{\rm{I}}^{\rm{R}16}}}\), where kI is the import rate for a protein of mass M and an unfolding force of FU, and where the import rate of the R16 monomer kIR16 has been used as reference. The dotted white line separates the regimes where mechanics or mass dominate the kinetics of nuclear import. h, Cartoon representation describing the passage of protein cargos across the NPC, in the absence (left) and presence (right) of a mechanically labile peptide tag. This results in the lowering of the free energy barrier and therefore in faster nuclear import kinetics. All points and bar plots indicate mean ± s.e.m.
