Single-molecule transport across an individual biomimetic nuclear pore complex

Journal name:
Nature Nanotechnology
Volume:
6,
Pages:
433–438
Year published:
DOI:
doi:10.1038/nnano.2011.88
Received
Accepted
Published online

Abstract

Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells1. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon2. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore3. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ~2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ~25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

At a glance

Figures

  1. Biomimetic NPC.
    Figure 1: Biomimetic NPC.

    a, Side-view schematic showing the device consisting of a 20 nm thin, free-standing silicon nitride window (blue layer) embedded in a silicon wafer (light green). A nanopore is drilled using a highly focused electron beam (yellow). b, Sketch showing the experimental concept. The biomimetic NPC is engineered by attaching FG-Nups to a solid-state nanopore, and transport of Impβ is measured by monitoring the trans-pore current. c, TEM images of the same nanopore with a diameter of 20 nm (top) or 40 nm (bottom) before (left) and after (right) coating with Nup98. d, Example of a current–voltage (I–V) curve before (red) and after (blue) coating a 40 nm nanopore with Nup98, showing an increased resistance due to the coating.

  2. Conductance measurements and models.
    Figure 2: Conductance measurements and models.

    a, Measured conductance versus pore diameter for bare pores (black points), Nup98-coated (green) and Nup153-coated (inset, red) pores. For all pores, the pore conductance decreases upon coating. Coloured lines are linear fits of two models (see text). Model 2 (solid lines) is found to fit the data much better than model 1 (dashed lines). bd, Schematics showing small- and large-pore regimes for models 1 and 2, as discussed in the text. Fitting to model 2 yields a Nup layer thickness tNup98 = 15 ± 1 nm and tNup153 = 8 ± 1 nm along the circumference of the pore for Nup98 and Nup153, respectively.

  3. Single-molecule translocation events.
    Figure 3: Single-molecule translocation events.

    a, Representative ion current trace before and after addition of Impβ in a bare pore. Downward spikes appear in the current trace upon addition of Impβ. Each spike is a single-molecule event. The lower panel shows zoom-ins on a number of events. b, As in a, but for a Nup98-coated pore. c, Scatter diagram for Impβ translocation in a bare (red) and Nup98-modified (black) pore, where each point represents an individual event. Event amplitudes are similar (~0.6 nS), but the dwell times differ by more than an order of magnitude (~200 µs versus ~3 ms). d, Scatter diagram for BSA translocation through bare (green) and Nup98 pores (black). e, As in c, but for a Nup153 pore. f, As in d, but for a Nup153-modified pore.

  4. Event frequencies through bare and Nup-modified pores, showing NPC-like selectivity.
    Figure 4: Event frequencies through bare and Nup-modified pores, showing NPC-like selectivity.

    Average number of events per second for BSA through a bare pore (green), Impβ through a bare pore (red), BSA through a Nup98-coated pore (blue), Impβ through a Nup98-coated pore (black), and finally BSA through a Nup153-coated pore (blue) and Impβ through a Nup153-coated pore (black). Pore diameter is 42–46 nm in all cases. The passage of BSA through the Nup-modified pore is significantly inhibited in Nup-coated pores, whereas that of Impβ is not; that is, these pores display the hallmark of NPC selectivity.

  5. Nanopore array.
    Figure 5: Nanopore array.

    a, TEM image of a nanopore array consisting of 61 pores with diameters of 43 ± 3 nm. b, TIRF image of individual Alexa488-labelled fluorescent Impβ proteins that were translocated through the Nup98-coated nanopore array of a to the trans chamber and subsequently immobilized onto a cover slide (see text). The inset shows a control image of buffer only, for exactly the same TIRF conditions.

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Author information

Affiliations

  1. Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands

    • Stefan W. Kowalczyk,
    • Timothy R. Blosser,
    • Tomás Magalhães,
    • Pauline van Nies &
    • Cees Dekker
  2. Biozentrum and the Swiss Nanoscience Institute, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

    • Larisa Kapinos &
    • Roderick Y. H. Lim

Contributions

S.W.K., R.Y.H.L. and C.D. devised the experiments. L.K. cloned, purified and labelled proteins and carried out SPR analysis. S.W.K., T.R.B., T.M. and P.V.N. carried out the experiments and analysed data. S.W.K., R.Y.H.L. and C.D. wrote the manuscript.

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The authors declare no competing financial interests.

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