Fig. 2: The mechano-directionality of the protein cargo determines its nuclear import kinetics across the NPC.
From: Structural anisotropy results in mechano-directional transport of proteins across nuclear pores
a, Unfolding forces (AFM force–extension, v = 400 nm s−1)51 of the soft spectrin R16 domain and stiff titin Ig27WT (WT). b, Heterodimer protein constructs alternatively placing the R16 or WT at the N-terminal position (after the NLS). c, Magnetic tweezers pulling experiments comparing the unfolding dynamics R16-WT (top) and WT-R16 (bottom) protein constructs. d–f, Nuclear import kinetics of heterodimers composed of the labile R16 domain, and an Ig27 variant (V13P, WT, Y9P) alternatively placed at the N- or C- terminus, to test the effect of protein mechano-directionality on the nuclear translocation kinetics. d, Representative confocal images of U2OS cells after 30 min into the recovery phase. Scale bar, 10 µm. e, Average time courses of the nucleus-to-cytoplasm localization of the respective protein construct. f, Import rates calculated from fits to the recovery time courses. R16-V13P (kI = 1.64 ± 0.04 ks−1, n = 276); V13P-R16 (kI = 1.72 ± 0.04 ks−1, n = 154); R16-WT (kI = 1.42 ± 0.04 ks−1, n = 192); WT-R16 (kI = 1.29 ± 0.04 ks−1, n = 260); R16-Y9P (kI = 1.58 ± 0.07 ks−1, n = 80); Y9P-R16 (kI = 1.02 ± 0.08 ks−1, n = 55). Significance levels for two-tailed the Mann–Whitney non-parametric test: NS, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. R16-V13P versus V13P-R16, P = 0.06; R16-WT versus WT-R16, P = 0.02; R16-Y9P versus Y9P-R16, P = 1.67 × 10−6. g, Relative directionality (calculated as the ratio between the import rates of the constructs with N-terminal R16 and C-terminal R16) compared to the difference in mechanical stability between each Ig27 variant and R16. h, Schematics highlighting the sensitivity of the NPC to the mechano-directionality of the translocating cargo. i, Unfolding forces (AFM force–extension, v = 400 nm s−1)55 measured for the titin Ig27WT (WT) and the pilin Spy0128 domain. Spy0128 is mechanically ultrastable; hence, WT plays in this case the role of the mechanically labile protein. j–l, Nuclear import kinetics of heteropolyproteins composed of two WT and one Spy0128 protein dimer. j, Representative confocal images of U2OS cells 30 min into the recovery phase. Scale bar, 10 µm. k,l, Average time courses of nucleus-to-cytoplasm localization (k) and associated import rates (l). (WT)2-Spy0128 (kI = 0.89 ± 0.07 ks−1, n = 70); WT-Spy0128-WT (kI = 0.99 ± 0.07 ks−1, n = 118); Spy0128-(WT)2 (kI = 0.56 ± 0.03 ks−1, n = 106). Significance levels for two-tailed Mann–Whitney non-parametric test: NS, P > 0.05; ****P < 0.0001. (WT)2-Spy0128 versus WT-Spy0128-WT, P = 0.80; WT-Spy0128-WT versus Spy0128-(WT)2, P = 5.70 × 10−5; (WT)2-Spy0128 versus Spy0128-(WT)2, P = 4.45 × 10−5. m, Schematics of the dynamics of nuclear translocation of Spy0128-(WT)2 versus (WT)2-Spy0128, highlighting the sensitivity of the NPC to the mechano-directionality of the shuttling protein cargo. All points and bar plots indicate mean ± s.e.m.
