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Anal Chem
2014 Feb 18;864:2090-8. doi: 10.1021/ac403607s.
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Ion permeability of the nuclear pore complex and ion-induced macromolecular permeation as studied by scanning electrochemical and fluorescence microscopy.
Kim J
,
Izadyar A
,
Shen M
,
Ishimatsu R
,
Amemiya S
.
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Efficient delivery of therapeutic macromolecules and nanomaterials into the nucleus is imperative for gene therapy and nanomedicine. Nucleocytoplasmic molecular transport, however, is tightly regulated by the nuclear pore complex (NPC) with the hydrophobic transport barriers based on phenylalanine and glycine repeats. Herein, we apply scanning electrochemical microscopy (SECM) to quantitatively study the permeability of the NPCs to small probe ions with a wide range of hydrophobicity as a measure of their hydrophobic interactions with the transport barriers. Amperometric detection of the redox-inactive probe ions is enabled by using the ion-selective SECM tips based on the micropipet- or nanopipet-supported interfaces between two immiscible electrolyte solutions. The remarkably high ion permeability of the NPCs is successfully measured by SECM and theoretically analyzed. This analysis demonstrates that the ion permeability of the NPCs is determined by the dimensions and density of the nanopores without a significant effect of the transport barriers on the transported ions. Importantly, the weak ion-barrier interactions become significant at sufficiently high concentrations of extremely hydrophobic ions, i.e., tetraphenylarsonium and perfluorobutylsulfonate, to permeabilize the NPCs to naturally impermeable macromolecules. Dependence of ion-induced permeabilization of the NPC on the pathway and mode of macromolecular transport is studied by using fluorescence microscopy to obtain deeper insights into the gating mechanism of the NPC as the basis of a new transport model.
Figure 1. (A) Scheme of the NPC
with central (red) and peripheral (blue)
barriers. The filaments and basket of the NPC are shown by wavy and
dotted lines, respectively. C and N represent cytoplasmic and nucleus
sides, respectively. NE is the nuclear envelope. (B) Cytoplasmic top
view (left) and side view (right) of the NPC with FG-rich nups forming
central (red) and peripheral (blue) barriers.
Figure 2. (A) Measurement illustration of the ion permeability of
the NE
using a micropipet-supported ITIES tip. The nucleus was swollen to
detach the NE from the nucleoplasm for smoothening. (B) SEM and (C)
FIB images of a milled micropipet. Scale bars, 1 μm.
Figure 3. Cyclic voltammograms of various ions at the
1,2-DCE/water interfaces
supported at ∼1 μm diameter pipets. Reference electrode,
Ag/AgCl. Potential sweep rate, 10 mV/s.
Figure 4. (A) Experimental approach curves for TPhAs+ at the NE
and the Si wafer as obtained with micropipet-supported ITIES tips.
Tip approach rate, 0.30 μm/s. (B) Plot of the ion permeability
of the NE against ion diffusion coefficient. The solid line is the
best fit of eq 2 with the experimental plot.
Figure 5. Approach curve at the NE as obtained using the
∼30 nm-diameter
pipet filled with the 1,2-DCE solution of the organic supporting electrolytes.
The external solution was the hypotonic buffer solution of 10 mM TBACl.
Tip approach rate, 60 nm/s.
Figure 6. Fluorescence
microscopic images of the whole nuclei in the isotonic
solution after incubation with (A) rhodamine-labeled BSA and (B) rhodamine-labeled
and NLS-tagged BSA and importins. In part (A), the nuclei were preincubated
with either TPhAs+ or PFBS– (left) and,
then, with the isotonic solution with (middle) or without (right)
WGA. In part (B), the nuclei were preincubated only with WGA (left)
or permeabilized by TPhAs+ (middle) and then washed in
the isotonic solution (right) before incubation with WGA.
Figure 7. Mechanism of
ion-induced permeabilization of (A) central mesh barriers
and (B) peripheral polymer-brush barriers to the importin-facilitated
transport of NLS-tagged BSA and the passive transport of BSA, respectively.
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