Artificial nanopores that mimic the transport selectivity of the nuclear pore complex

Abstract

Nuclear pore complexes (NPCs) act as effective and robust gateways between the nucleus and the cytoplasm, selecting for the passage of particular macromolecules across the nuclear envelope. NPCs comprise an elaborate scaffold that defines a approximately 30 nm diameter passageway connecting the nucleus and the cytoplasm. This scaffold anchors proteins termed 'phenylalanine-glycine' (FG)-nucleoporins, the natively disordered domains of which line the passageway and extend into its lumen. Passive diffusion through this lined passageway is hindered in a size-dependent manner. However, transport factors and their cargo-bound complexes overcome this restriction by transient binding to the FG-nucleoporins. To test whether a simple passageway and a lining of transport-factor-binding FG-nucleoporins are sufficient for selective transport, we designed a functionalized membrane that incorporates just these two elements. Here we demonstrate that this membrane functions as a nanoselective filter, efficiently passing transport factors and transport-factor-cargo complexes that specifically bind FG-nucleoporins, while significantly inhibiting the passage of proteins that do not. This inhibition is greatly enhanced when transport factor is present. Determinants of selectivity include the passageway diameter, the length of the nanopore region coated with FG-nucleoporins, the binding strength to FG-nucleoporins, and the antagonistic effect of transport factors on the passage of proteins that do not specifically bind FG-nucleoporins. We show that this artificial system faithfully reproduces key features of trafficking through the NPC, including transport-factor-mediated cargo import.

Figure 1. Design and operation of the NPC mimic

a, Far left: schematic of a single pore in the functionalized membrane. A 6-μm-thick polycarbonate membrane perforated by ∼30-nm channels (8 × 10⁸ pores per cm²) was coated on one face with a ∼15-nm-thick gold layer to which we attached FG-nucleoporins (FG-nups) by a single carboxy-terminal cysteine; hence, all FG-nucleoporins were similarly oriented. Subsequent attachment of 356-Da PEG-thiol (small PEG) molecules blocked any remaining exposed gold surface,. Centre left: exploded view of the device carrying the membrane. Centre right: sectional view of the device mounted on a confocal microscope and loaded with a fluorescently labelled protein solution (green). Lower chamber: height, ∼25 μm; diameter, ∼1.6 mm. Upper chamber: height, ∼1 mm; diameter, ∼2 mm; overflow capacity, 100 μl. Far right: top-view photograph of the device. b, Transmission electron micrograph of a single pore on Nsp1FG-functionalized membrane, incubated with gold-labelled NTF2–GST (pseudocoloured red). NTF2–GST is seen to bind the FG-nucleoporin layer projecting from the gold surface and transit into the 30-nm pore. c, Method for measurement of protein fluxes across the nano-selective filter. Left: confocal microscopy z-axis section through the lower chamber, membrane and upper chamber showing the fluorescence signal (blue) from a single protein in equilibrium between the two chambers. The measured volume is indicated by the dashed box. Right: plot showing the decrease in fluorescence signal in the lower chamber after dilution of the upper chamber, resulting from an efflux of protein from the lower to upper chamber (Supplementary Information). d, Two-channel fluorescence measurements of simultaneous diffusion of fluorescein isothiocyanate (FITC)-labelled BSA (blue) and Cy5-labelled NTF2–GST (red) through either an Nsp1FG-coated membrane (top) or a control small-PEG-coated membrane (bottom). Left: time course of confocal images collected as in c. Colours were altered for clarity; the same data with unmodified colours are provided in Supplementary Fig. 12. Right: corresponding fluorescence decrease curves and fluxes (in molecules pore⁻¹ s⁻¹ μM⁻¹). The flux of NTF2–GST was similar through both membranes, whereas the flux of BSA was significantly lower through just the Nsp1FG-coated membrane.

Figure 2. Selective trafficking of transport factors and cargo through the nanopores

A flux ratio was obtained for each protein by calculating the ratio of its flux through a functionalized membrane (in this case Nsp1FG) relative to its flux through the control small PEG membrane. A value of 1 indicates no reduction in flux through the functionalized membrane as compared with the control membrane, whereas a value of 0 indicates no flux through the functionalized membrane. a, Flux ratios of NTF2–GST versus control proteins. Flux ratios plotted against Stokes radius for variously sized proteins in the presence of NTF2–GST (R_s = 3.6 nm), namely: RNase A (1.75 nm), GFP (2.8 nm), BSA (3.5 nm), transferrin (3.6 nm), immunoglobulin G (IgG; 5.1 nm). The flux ratio of all proteins except NTF2–GST was reduced in a size-dependent manner. b, Flux ratios of two karyopherins versus control protein (BSA). Flux ratios for a mixture of BSA and Kap95 and a mixture of BSA and Kap121 showed BSA having a markedly lower flux ratio than either karyopherin (despite the smaller size of BSA) in a manner similar to a mixture of BSA and NTF2–GST. c, Karyopherin-mediated transport of cargo. Flux ratios for either GFP or Ibb–GFP in the presence of Kap95 (not fluorescently labelled) and IgG; Ibb–GFP forms a complex with Kap95, whereas GFP does not (see Supplementary Information). The flux ratio of Ibb–GFP in a complex with Kap95 was higher than that of either GFP alone (even though GFP has a much smaller Stokes radius than the Ibb–GFP/Kap95 complex) or IgG alone. Hence, the device mimics Kap-mediated cargo transport. Standard errors are shown.

Figure 3. Presence of transport factor enhances the selectivity of FG-nucleoporin-coated membranes

a, Left, time course of confocal images collected as in Fig. 1, comparing fluxes of BSA through the small PEG membrane (bottom), the Nsp1FG membrane (middle), and the Nsp1FG membrane in the presence of NTF2–GST (top); right, corresponding fluorescence decrease curves and fluxes (in molecules pore⁻¹ s⁻¹ μM⁻¹). b, Strong selectivity of NTF2–GST transport over BSA is observed for the Nsp1FG functionalized membrane and is not observed for the ‘inert’ 30 kDa PEG functionalized membrane. Flux ratios are plotted for BSA and NTF2–GST alone (1 species) or in combination with each other (2 species), through either Nsp1FG or 30 kDa PEG membranes versus small PEG membrane. Standard errors are shown.

Figure 4. The effect of pore geometry and FG-nucleoporin binding strength on transport

a, The selectivity of the Nsp1FG membranes decreases with increasing pore diameter. Flux ratio measurements of a mixture of BSA and NTF2–GST were performed as in Fig. 2a, for membranes having differing pore diameters. b, The selectivity of the Nsp1FG membrane was significantly improved when the thickness of the gold layer was increased. c, In vitro, NTF2(WT)–GST (red) had a lower apparent K_d value (10 ± 3 nM) than mutant NTF2(W7A)–GST (pink, K_d of 21 ± 4 nM) for binding to Nsp1FG, and at the same time had about double the binding capacity compared to the mutant. d, The NTF2(W7A)–GST mutant had a reduced flux ratio compared to NTF2(WT)–GST (for flux through the Nsp1FG membrane, P = 0.009, single-tailed t-test), although no flux difference was seen on the control small-PEG-coated membranes (P = 0.35). e, Assessment of the effect on transport of binding strength to FG-nucleoporins by changing the FG-repeat motif. Flux ratios through either Nsp1FG or Nup100FG membranes are shown for the Kap95/Ibb–GFP complex with IgG and for NTF2–YFP with BSA. Because NTF2 binds poorly to Nup100FG compared to its binding strength to Nsp1FG, Nup100FG-coated membranes did not discriminate well between BSA and NTF2–YFP. Standard errors are shown in parts a, b, d and e, and standard deviations are shown in c.