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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 â¼1 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
â¼30 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 PFBSâ (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.
Akita,
Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose.
2011, Pubmed
Akita,
Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose.
2011,
Pubmed
Amemiya,
Scanning electrochemical microscopy.
2008,
Pubmed
Amemiya,
Scanning electrochemical microscopy. 40. Voltammetric ion-selective micropipet electrodes for probing ion transfer at bilayer lipid membranes.
2000,
Pubmed
Bird,
Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.
2001,
Pubmed
Boika,
Monitoring the electrophoretic migration and adsorption of single insulating nanoparticles at ultramicroelectrodes.
2013,
Pubmed
Chatel,
Domain topology of nucleoporin Nup98 within the nuclear pore complex.
2012,
Pubmed
,
Xenbase
Duverger,
Nuclear import of glycoconjugates is distinct from the classical NLS pathway.
1995,
Pubmed
Eisele,
Cohesiveness tunes assembly and morphology of FG nucleoporin domain meshworks - Implications for nuclear pore permeability.
2013,
Pubmed
Elsamadisi,
Polished nanopipets: new probes for high-resolution scanning electrochemical microscopy.
2011,
Pubmed
Fajac,
Targeting of cell receptors and gene transfer efficiency: a balancing act.
2002,
Pubmed
Falces,
Recognition of nucleoplasmin by its nuclear transport receptor importin α/β: insights into a complete import complex.
2010,
Pubmed
,
Xenbase
Finlay,
Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
1987,
Pubmed
,
Xenbase
Frenkiel-Krispin,
Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture.
2010,
Pubmed
,
Xenbase
Frey,
FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties.
2006,
Pubmed
Frey,
A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes.
2007,
Pubmed
Görlich,
Transport between the cell nucleus and the cytoplasm.
1999,
Pubmed
Guo,
Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy.
2005,
Pubmed
,
Xenbase
Herrmann,
Near-field optical study of protein transport kinetics at a single nuclear pore.
2009,
Pubmed
,
Xenbase
Hoelz,
The structure of the nuclear pore complex.
2011,
Pubmed
Hülsmann,
The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model.
2012,
Pubmed
,
Xenbase
Ishimatsu,
Electrochemical mechanism of ion-ionophore recognition at plasticized polymer membrane/water interfaces.
2011,
Pubmed
Ishimatsu,
Ion-selective permeability of an ultrathin nanoporous silicon membrane as probed by scanning electrochemical microscopy using micropipet-supported ITIES tips.
2010,
Pubmed
Jing,
High lipophilicity of perfluoroalkyl carboxylate and sulfonate: implications for their membrane permeability.
2009,
Pubmed
Kim,
Nanoscale mechanism of molecular transport through the nuclear pore complex as studied by scanning electrochemical microscopy.
2013,
Pubmed
Kim,
A structure-permeability relationship of ultrathin nanoporous silicon membrane: a comparison with the nuclear envelope.
2008,
Pubmed
Kim,
Stabilizing nanometer scale tip-to-substrate gaps in scanning electrochemical microscopy using an isothermal chamber for thermal drift suppression.
2012,
Pubmed
Kirschner,
Characterization of the nuclear envelope, pore complexes, and dense lamina of mouse liver nuclei by high resolution scanning electron microscopy.
1977,
Pubmed
Labokha,
Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes.
2013,
Pubmed
,
Xenbase
Liashkovich,
Structural organization of the nuclear pore permeability barrier.
2012,
Pubmed
,
Xenbase
Lim,
Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport.
2006,
Pubmed
Löschberger,
Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.
2012,
Pubmed
,
Xenbase
Ma,
Self-regulated viscous channel in the nuclear pore complex.
2012,
Pubmed
Meyerhoff,
Polyion-sensitive membrane electrodes for biomedical analysis.
1996,
Pubmed
Milles,
Facilitated aggregation of FG nucleoporins under molecular crowding conditions.
2013,
Pubmed
Naim,
Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier.
2009,
Pubmed
Nioradze,
Origins of nanoscale damage to glass-sealed platinum electrodes with submicrometer and nanometer size.
2013,
Pubmed
Opferman,
Morphology of polymer brushes infiltrated by attractive nanoinclusions of various sizes.
2013,
Pubmed
Opferman,
Morphological control of grafted polymer films via attraction to small nanoparticle inclusions.
2012,
Pubmed
Pack,
Design and development of polymers for gene delivery.
2005,
Pubmed
Powers,
The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways.
1997,
Pubmed
,
Xenbase
Quinn,
Time-resolved electrochemical detection of discrete adsorption events.
2004,
Pubmed
Raices,
Nuclear pore complex composition: a new regulator of tissue-specific and developmental functions.
2012,
Pubmed
Ren,
Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1.
2010,
Pubmed
Ribbeck,
The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.
2002,
Pubmed
Schoch,
Nuclear transport receptor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes.
2012,
Pubmed
Schwefel,
Structural basis of multivalent binding to wheat germ agglutinin.
2010,
Pubmed
Shahin,
Investigation of nuclear envelope structure and passive permeability.
2009,
Pubmed
,
Xenbase
Shen,
Quantitative imaging of ion transport through single nanopores by high-resolution scanning electrochemical microscopy.
2012,
Pubmed
Solmaz,
Ring cycle for dilating and constricting the nuclear pore.
2013,
Pubmed
Strambio-De-Castillia,
The nuclear pore complex: bridging nuclear transport and gene regulation.
2010,
Pubmed
Terry,
Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport.
2007,
Pubmed
Wang,
Conformational changes of the in situ nuclear pore complex.
1999,
Pubmed
,
Xenbase
Wang,
Resistive-pulse measurements with nanopipettes: detection of Au nanoparticles and nanoparticle-bound anti-peanut IgY.
2013,
Pubmed
Yamada,
A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins.
2010,
Pubmed
Zhan,
The Kv channel blocker 4-aminopyridine enhances Ag+ uptake: a scanning electrochemical microscopy study of single living cells.
2008,
Pubmed
Zheng,
Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation.
2012,
Pubmed
