Karyopherins regulate nuclear pore complex barrier and transport function

Kapinos, Larisa E.; Huang, Biao; Rencurel, Chantal; Lim, Roderick Y. H.

doi:10.1083/jcb.201702092

Cited by 89 publications

(127 citation statements)

References 78 publications

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“…We fitted a single Langmuir isotherm to the saturation points, see Figure 2c, and thus obtained a binding affinity of -= 191 ± 20 nM (error is S.D.). This value compares very well tovalues for the binding between Kap95 and native FG-Nups as found with similar techniques, which typically range between 100 and 500 nM 18,27 . For reference, we similarly measured the affinity of Kap95 to Nsp1, a native FG-Nup from yeast, as well as to Nsp1-S, a Nsp1-mutant where the hydrophobic amino acids F, I, L, V are replaced by the hydrophilic amino acid Serine (S), which serves as a negative control since it is expected to not bind Kap95 due to the lack of FG repeats 14,15 (Figure 2d,g).…”

Section: Qcm-d Experiments and MD Simulations Show Selective Bindingsupporting

confidence: 81%

“…That the Kap95 to NupX and Nsp1(-S) binding constants were found using different fits (single component vs. two-component Langmuir isotherms, respectively) can be attributed to differences between NupX and Nsp1 in terms of their respective ratios of ' and : . In previous work 18,46 , sparsely grafted FG domains ( ' > : ) revealed a single type of binding when interacting with Impβ (Kap95 human homolog) molecules, as opposed to closely-packed FG domains ( ' < : ) that instead showed two binding modes. Following this reasoning, the use of a two-component isotherm for the Nsp1(-S) brushes would be consistent with these and other previous studies 17,18 , since : (7.9 ± 2.0 nm for Nsp1, 6.8 ± 1.6 nm for Nsp1-S; errors are S.D.…”

Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning

confidence: 96%

“…Assuming 35% of the sensed film to be constituted by protein mass 43 , with the remaining 65% being constituted by coupled water, we estimated an average grafting distance ' between adjacent NupX proteins of '~5 .6 nm, assuming a triangular lattice (since an equilateral triangulated (hexagonal) lattice is the densest type of packing that can be described by a unique length scale that sets the grafting density). After the Nup-layer was formed, a 1-mercapto-11undecylte-tra(ethyleneglycol) molecule, which is expected to form a ~2 nm thin passivating film 17 , was added to passivate any remaining bare gold that was exposed in between NupX molecules, which minimizes unintentional interactions between Kap95 and gold ( Figure S3) 17,18,45 . After thus setting up a NupX-coated layer, we flushed in Kap95 at stepwise increasing concentrations (~10-1000 nM, Figure 2b) and monitored binding to the NupX-coated surface.…”

Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning

confidence: 99%

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A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Fragasso

Vries

Sluis

et al. 2020

Preprint

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Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure a strong affinity of the artificial FG-Nup brushes to the transport receptor Kap95, whereas no binding occurs to cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create functional NPCs. IntroductionNucleocytoplasmic transport is orchestrated by the Nuclear Pore Complex (NPC), which imparts a selective barrier to biomolecules 1,2 . The NPC is a large eightfold-symmetric protein complex (with a size of ~52 MDa in yeast and ~112 MDa in vertebrates) that is embedded within the nuclear envelope and comprises ~30 different types of Nucleoporins ('Nups') 3,4 . Intrinsically disordered proteins, termed FG-Nups, line the central channel of the NPC. FG-Nups are characterized by the presence of phenylalanine-glycine (FG) repeats separated by spacer sequences 5 and they are highly conserved throughout species 6 . FG-Nups carry out a dual function: By forming a dense barrier (100-200 mg/mL) within the NPC lumen, they allow passage of molecules in a size-selective manner 7-10 . Small molecules can freely diffuse through, whereas larger particles are generally excluded 11 . At the same time, FG-Nups mediate the transport of large NTR-bound (Nuclear Transport Receptor) cargoes across the NPC through transient hydrophobic interactions between FG repeats and hydrophobic pockets on the convex side of NTRs 12 . Various models have been developed in order to connect the physical properties of FG-Nups to the size-selective properties of the NPC central channel, e.g. the 'virtual-gate' 13 , 'selective phase' 14,15 , 'reduction of dimensionality' 16 , 'kap-centric' 17-19 , 'polymer brush' 20 , and 'forest' 5 models.As is evident from the multitude of transport models, no consensus on the NPC transport mechanisms has yet been reached.The NPC is highly complex in its architecture and dynamics, being constituted by many different Nups that simultaneously interact with multiple transiting cargoes and NTRs. In fact, translocating cargoes may amount to almost half of the mass of the central channel, so they may be considered an ...

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Section: Qcm-d Experiments and MD Simulations Show Selective Bindingsupporting

confidence: 81%

Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning

confidence: 96%

Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning

confidence: 99%

See 1 more Smart Citation

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Fragasso

Vries

Sluis

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Those might also enable a quantitative comparison between the permeability barrier properties of hydrogels versus liquid droplets under same conditions. Recent studies have suggested an integral role of NTRs as part of the permeability barrier (Kapinos et al, 2017; Lowe et al, 2015). Microrheology measurements could also investigate the permeability barrier properties over material states to assay if the presence of NTRs affect the molecular architecture of the droplet.…”

Section: Resultsmentioning

confidence: 99%

The liquid state of FG-nucleoporins mimics permeability barrier properties of nuclear pore complexes

Celetti

Paci

Caria

et al. 2019

Journal of Cell Biology

106

116

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Nuclear pore complexes (NPCs) regulate all cargo traffic across the nuclear envelope. The transport conduit of NPCs is highly enriched in disordered phenylalanine/glycine-rich nucleoporins (FG-Nups), which form a permeability barrier of still elusive and highly debated molecular structure. Here we present a microfluidic device that triggered liquid-to-liquid phase separation of FG-Nups, which yielded droplets that showed typical properties of a liquid state. On the microfluidic chip, droplets were perfused with different transport-competent or -incompetent cargo complexes, and then the permeability barrier properties of the droplets were optically interrogated. We show that the liquid state mimics permeability barrier properties of the physiological nuclear transport pathway in intact NPCs in cells: that is, inert cargoes ranging from small proteins to large capsids were excluded from liquid FG-Nup droplets, but functional import complexes underwent facilitated import into droplets. Collectively, these data provide an experimental model of how NPCs can facilitate fast passage of cargoes across an order of magnitude in cargo size.

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“…Mediates NLS-NC-FG Nup Interactions. Multivalent interactions between Kaps and FG Nups facilitate selective transport across the NPC (40). We ascertained the binding of Kapα•Kapβ1•NLS-NCs to three FG Nups (cNup98, cNup214, and cNup153) by surface plasmon resonance (SPR) (SI Appendix, Fig.…”

Section: Kapα•kapβ1mentioning

confidence: 99%

Organelle-specific targeting of polymersomes into the cell nucleus

Zelmer

Zweifel

Kapinos

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

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Karyopherins regulate nuclear pore complex barrier and transport function

Cited by 89 publications

References 78 publications

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

The liquid state of FG-nucleoporins mimics permeability barrier properties of nuclear pore complexes

Organelle-specific targeting of polymersomes into the cell nucleus

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