Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors

Eisele, Nico B.; Frey, Steffen; Piehler, Jacob; Görlich, Dirk; Richter, Ralf P.

doi:10.1038/embor.2010.34

Cited by 106 publications

(204 citation statements)

References 23 publications

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“…This is supported by our observation that yeast GLFG domains can be compacted into the NPC or extended. Eisele et al (Eisele et al, 2010) showed, using atomic force microscopy, that dense FG domain films (with a similar density to the channel) did not collapse in the presence of importin b, but less densely packed ones could (Lim et al, 2007). This is consistent with the suggestion that some GLFG domains are densely packed in the channel and hence might not undergo reversible collapse (Eisele et al, 2010), but others can exist in extended, but reversibly collapsible conformations (Lim et al, 2007).…”

Section: Discussionsupporting

confidence: 74%

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains

Fišerová

Spink

Richards

et al. 2013

Journal of Cell Science

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Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycineleucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction. We investigated this possibility during nuclear transport. Using electron microscopy of S. cerevisiae, we show that NPC cytoplasmic filaments form a dome-shaped structure enclosing GLFG domains. GLFG domains extend out of this structure and are part of an 'exclusion zone' that might act as a partial barrier to entry of transport-inert proteins. The anchor domain of a GLFG nucleoporin locates exclusively to the central channel. By contrast, the localisation of the GLFG domains varied between NPCs and could be cytoplasmic, central or nucleoplasmic and could stretch up to 80 nm. These results suggest a dynamic exchange between ordered and disordered states. In contrast to diffusion through the NPC, transport cargoes passed through the exclusion zone and accumulated near the central plane. We also show that movement of cargo through the NPC is accompanied by relocation of GLFG domains, suggesting that binding, restructuring and movement of these domains could be part of the translocation mechanism.

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Section: Discussionsupporting

confidence: 74%

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains

Fišerová

Spink

Richards

et al. 2013

Journal of Cell Science

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“…43,44 Similarly, FG-domains were used to coat supported lipid bilayers and to produce a densely attached ultrathin FG-domain film, to which the NTR could also bind. 67 The evidence from these studies supports the selective phase/hydrogel proposal. The cohesive FGNups include Nup42, Nup49, Nup57, Nup100, Nup116, and Nup145N.…”

Section: Distinct Transport Routes and Proposed Transport Modelssupporting

confidence: 71%

The selective permeability barrier in the nuclear pore complex

Goryaynov

Yang

2016

Nucleus

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The nuclear pore complex (NPC) mediates the shuttle transport of macromolecules between the nucleus and cytoplasm in eukaryotic cells. The permeability barrier formed by intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-Nups) in the NPC functions as the critical selective control for nucleocytoplasmic transport. Signal-independent small molecules (< 40 kDa) passively diffuse through the pore, but passage of large cargo molecules is inhibited unless they are chaperoned by nuclear transport receptors (NTRs). NTRs are capable of interacting with FG-Nups and guide the cargos to cross the barrier by facilitated diffusion. The native conformation of the FGNups permeability barrier and the competition among multiple NTRs interacting with this barrier in the native NPCs are the 2 core questions still being highly debated in the field. Recently, we applied high-speed super-resolution fluorescence microscopy to map out the natural structure of the FGNups barrier and determined the competition among multiple NTRs as they interact with the barrier in the native NPCs. In this extra-view article, we will review the current understanding in the configuration and function of FG-Nups barrier and highlight the new evidence obtained recently to answer the core questions in nucleocytoplasmic transport.

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“…The bulk-like hydrogel meshwork model (5), for instance, places a greater importance on the hydrophobic interactions between neighbor FG-Nups, as they are able to form a sieve-like meshwork in vitro (10). Transport could then occur through binding of transport receptors to FG-repeats, causing a local gel-to-liquid transition and allowing the receptor to catalyze its own diffusion (28). Our data do not exclude that these interactions play a role in transport; however, they suggest that the most kinetically relevant events in the intact NPC have the characteristics of directed transport, not of unbiased diffusion.…”

Section: Resultsmentioning

confidence: 99%

Capturing directed molecular motion in the nuclear pore complex of live cells

Cardarelli

Lanzanò

Gratton

2012

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

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Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinβ1 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs.fluctuation spectroscopy | particle tracking N uclear pore complexes (NPCs) regulate the exchange of macromolecules between the nucleus and the cytoplasm (1, 2). Each eukaryotic NPC is an approximately 120-MDa supramolecular complex of about 30 different polypeptides designated nucleoporins (Nups) (3) approximately one-third of which are rich in natively unfolded Phe-Gly (FG) repeat domains (4). They form a selective filter inside the NPC that inhibits the efficient translocation of large cargo molecules (>40 kD) (5), unless they are chaperoned by transport receptors known as karyopherins (Kaps; also called importins and exportins) (6). In spite of the progress made in understanding NPC structure and its implications for nucleocytoplasmic transport, several aspects of how individual NPCs facilitate cargo translocation remain unresolved and represent a formidable challenge in present research. Such uncertainties stem from the complex nature of NPCs and from limitations associated with the current experimental approaches used to probe the NPC mechanism at the nanomolecular level and under real-time trafficking conditions. One of the controversies surrounding the field involves the origin of the "paradoxical" selective gating mechanism, which remains largely unknown. Different figurative translocation models have been proposed so far (for a review see ref.2). In the "virtual gating" model (7), the NPC allocates a large channel decorated at both the cytoplasmic and nucleoplasmic entrances with several FG-Nup fibrils. In this scheme, flexible and largely unstructured FG-domains limit available space in the NPC near field, thus restricting access of nontransport substrates to the channel. Conversely, FG-Nups increase the residence time of transport complexes in the central aperture of the pore by binding to nuclear transport receptors. In this way, FG-Nups facilitate diffusion of transport complexes into the central channel. In the "affinity gradient" model (8), FG-Nups are arranged along the NPC channel that their affinity for FG-binding molecules increases along the NPC axis. FG-binding molecules ...

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Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors

Cited by 106 publications

References 23 publications

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains

The selective permeability barrier in the nuclear pore complex

Capturing directed molecular motion in the nuclear pore complex of live cells

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