Nanoscale mechanism of molecular transport through the nuclear pore complex as studied by scanning electrochemical microscopy

Abstract

The nuclear pore complex (NPC) is the proteinaceous nanopore that solely mediates molecular transport across the nuclear envelope between the nucleus and cytoplasm of a eukaryotic cell. Small molecules (<40 kDa) diffuse through the large pore of this multiprotein complex. A passively impermeable macromolecule tagged with a signal peptide is chaperoned through the nanopore by nuclear transport receptors (e.g., importins) owing to their interactions with barrier-forming proteins. Presently, this bimodal transport mechanism is not well understood and is described by controversial models. Herein, we report on a dynamic and spatially resolved mechanism for NPC-mediated molecular transport through nanoscale central and peripheral routes with distinct permeabilities. Specifically, we develop a nanogap-based approach of scanning electrochemical microscopy to precisely measure the extremely high permeability of the nuclear envelope to a small probe molecule, (ferrocenylmethyl)trimethylammonium. Effective medium theories indicate that the passive permeability of 5.9 × 10(-2) cm/s corresponds to the free diffusion of the probe molecule through ~22 nanopores with a radius of 24 nm and a length of 35 nm. Peripheral routes are blocked by wheat germ agglutinin to yield 2-fold lower permeability for 17 nm-radius central routes. This lectin is also used in fluorescence assays to find that importins facilitate the transport of signal-tagged albumin mainly through the 7 nm-thick peripheral route rather than through the sufficiently large central route. We propose that this spatial selectivity is regulated by the conformational changes in barrier-forming proteins that transiently and locally expand the impermeably thin peripheral route while blocking the central route.

Figure 1

(A) The NPC with a barrier region (green), cytoplasmic filaments (wavy line), and a nuclear basket (dotted line) embedded in the NE. C and N represent the cytoplasmic and nucleus sides, respectively. (B–D) Top and side views of the barrier region with cohesive (green meshes) and non-cohesive (red wavy lines) FG domains (see the main text for the corresponding models).

Figure 2

SECM-induced transfer mode for the measurement of passive NE permeability to FcTMA⁺. The blue circles represent the oxidized form of FcTMA⁺.

Figure 3

(A) The SECM cell with a swollen nucleus. (B) Video microscopic image of the NE in contact with the top Si₃N₄ membrane of the cell.

Figure 4

Video microscopic images of a FIB-milled Pt tip positioned (A) above and (B) in the 10 μm × 10 μm opening of the SECM cell. (C) Approach curves at the NE in the hypotonic MIB solution of 0.3 mM FcTMA⁺ with and without 1.0 g/L WGA. Tip potential, 0.55 V vs. Ag/AgCl. Tip approach rate, 0.30 μm/s.

Figure 5

(A) Cross section of the concentration profile of FcTMA⁺ around the tip–NE nanogap as simulated by the finite element method with d/a = 0.3 in Figure S-5A. (B) Experimental and simulated approach curves at the NE with and without 1.0 g/L WGA. The respective simulation curves employed a = 0.44 μm and 0.42 μm with RG = 2.5. The theoretical negative approach curve was calculated for RG = 2.5.

Figure 6

Three-step diffusion of FcTMA⁺ through the NPC in the (A) absence and (B) presence of WGA. Each step is explained in the text.

Figure 7

Fluorescence microscopic images of swollen nuclei in the hypotonic MIB solution of rhodamine-labeled and NLS-tagged BSA (A) without and (B and C) with importins and energy mix. In part (C), the nucleus was incubated with 1.0 g/L WGA before the fluorescence transport assay.

Figure 8

Side and top views of the barrier region of the NPC based on the forest model. Green meshes represent cohesive FG domains. In part (B), the central transport barriers are composed of the FG-rich nups of the Xenopus NPC. Nup98 is anchored to the pore wall through Nup214 (not shown).