Near-Field Optical Study of Protein Transport Kinetics at a Single Nuclear Pore

Herrmann, Michael; Neuberth, Nicole; Wissler, Jörg; Pérez, J. Franco; Gradl, Dietmar; Naber, A.

doi:10.1021/nl901598z

Cited by 45 publications

(37 citation statements)

References 44 publications

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“…Previous controversial experimental results have fueled a debate over the spatial and functional overlap between passive and facilitated modes of transport (24)(25)(26)(27)(28). In our measurements, compared to cargo-free Imp β1, the import cargo complex pervaded more into the central passive diffusion pathway (the diameter of the Imp β1-unoccupied central channel in the middle plane of the NPC was found to be 23 AE 3 nm with cargo-free Imp β1 and only 8 AE 4 nm with cargo-bound Imp β1, Fig.…”

Section: Distinct But Not Completely Separate Passive and Facilitatedmentioning

confidence: 99%

Self-regulated viscous channel in the nuclear pore complex

Goryaynov

Sarma

et al. 2012

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

129

232

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The nuclear pore complex (NPC), the sole gateway for nucleocytoplasmic exchange in eukaryotic cells, allows for the passive diffusion of small molecules and transport-receptor-facilitated translocation of signal-dependent cargo molecules. Whether small molecules passively diffuse through a single central channel or through multiple holes of a hydrogel network is a subject of debate. Additionally, whether the passive and facilitated transport systems occupy distinct or overlapping physical regions of the NPC remains unclear. Here, we directly test these models using threedimensional super-resolution fluorescence microscopy of human cells. This approach reveals that a single viscous central channel in the NPC acts as the sole pathway for passive diffusion of various small molecules; transport receptors and their cargo complexes take distinct transport routes in the periphery, which is occluded by phenylalanine-glycine filaments. Furthermore, the passive and facilitated passageways in the NPC are closely correlated, and their conformations can be simultaneously regulated by Importin β1 (a major transport receptor) and RanGTP (a critical regulator of transport directionality). These results strongly favor a self-regulated viscous channel configuration in native NPCs over the porous hydrogel meshwork model. 3D imaging | nucleoporins | single-molecule fluorescence | super-resolution microscopy T he nuclear pore complex (NPC) that is embedded in the nuclear envelope (NE) functions as a highly selective gateway for an exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. The NPC is a large assembly of approximately 30 different proteins, known as nucleoporins (Nups), with each present in an integer multiple of eight copies (1-3). Approximately one-third of the total Nups possess a "natively unfolded" structure with domains that are rich in phenylalanineglycine (FG) repeats. These FG-Nups form the selective permeability barrier in the NPC that allows for two transport modes: passive diffusion of small molecules (<40-60 kDa) and transport-receptor-facilitated translocation of cargo molecules containing specific signals (4, 5). The existence of facilitated translocation suggests that a cargo molecule would be repelled by the NPC unless it is chaperoned by transport receptors (6-8).The dissociation and association of transport-receptor-cargo complexes are guided by a concentration gradient of RanGTP (or RanGDP) across the NE (9, 10). In contrast, the passive diffusion through the NPC requires no consumption of chemical energy.Electron microscopy studies have revealed that the central nuclear pore is approximately 40-90 nm in length, with a minimum internal diameter of around 40-75 nm and an external diameter of approximately 120 nm. Flexible filaments extend approximately 50 nm into the cytoplasm, and a basket structure extends approximately 75 nm into the nucleus (5, 11). Thus, a transiting substrate can potentially interact continuously with Nups over a distance spanning approximately 200 nm. N...

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Section: Distinct But Not Completely Separate Passive and Facilitatedmentioning

confidence: 99%

Self-regulated viscous channel in the nuclear pore complex

Goryaynov

Sarma

et al. 2012

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

129

232

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“…What is the degree of hydrophobicity required to define a specific Kap-FG interaction (in light of recent evidence showing that a degree of hydrophobic character is needed for non-specific translocation to proceed; Naim et al, 2009)? Is the observed Brownian motion of Kap-cargo complexes within the NPC (Herrmann et al, 2009) dependent on FG-domain behavior? How does a Kap slide and can Kap-sliding be promoted along a collapsed FG-domain surface?…”

Section: Discussionmentioning

confidence: 99%

“…Once past the FG-domain 'corona' or 'cloud', a Kap-cargo complex could exhibit unhindered Brownian motion within the very middle of the central channel (Yang et al, 2004;Herrmann et al, 2009).…”

Section: Fg-domains In the Npcmentioning

confidence: 99%

Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex

Peleg

Lim

2010

Biological Chemistry

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Several biological mechanisms involve proteins or proteinaceous components that are intrinsically disordered. A case in point pertains to the nuclear pore complex (NPC), which regulates molecular transport between the nucleus and the cytoplasm. NPC functionality is dependent on unfolded domains rich in Phe-Gly (FG) repeats (i.e., FG-domains) that collectively act to promote or hinder cargo translocation. To a large extent, our understanding of FG-domain behavior is limited to in vitro investigations given the difficulty to resolve them directly in the NPC. Nevertheless, recent findings indicate a collective convergence towards rationalizing FG-domain function. This review aims to glean further insight into this fascinating problem by taking an objective look at the boundary conditions and contextual details underpinning FG-domain behavior in the NPC. Here, we treat the FG-domains as being commensurate with polymeric chains to address ambiguities such as for instance, how FG-domains tethered to the central channel of the NPC would behave differently as compared with their free-floating counterparts in solution. By bringing such fundamental questions to the fore, this review seeks to illuminate the importance of how such parameters can hold influence over the structure-function relation of intrinsically disordered proteins in the NPC and beyond.

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“…Above this threshold, molecules are ushered selectively by dedicated transport receptors, which recognize specific nuclear localization (NLS) or nuclear export signal (NES) peptides displayed by the cargo (Pemberton and Paschal 2005). Molecular transport across the NE has been quantitatively addressed by the classical SPT approach, with the calculation of passive/active translocation times and trajectories at the single-molecule and single-pore level in permeabilized or microinjected cells (Yang et al 2004;Kubitscheck et al 2005;Dange et al 2008;Herrmann et al 2009;Ma and Yang 2010). Here, we will show how similar results can be obtained using GFP, in unperturbed cells, and in the presence of many molecules.…”

Section: Molecular Flow Across Obvious Barriers: the Border Of Non-comentioning

confidence: 99%

Measuring the flow of molecules in cells

Hinde

Cardarelli

2011

Biophys Rev

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No methods proposed thus far have the capability to measure molecular flow in live cells at the single molecule level. Here, we review the potentiality of a newly established method based on the spatial correlation of fluorescence fluctuations at a pair of points in the sample (pair correlation method). The pair correlation function (pCF) offers a unique tool to probe the directionality of intracellular traffic, by measuring the accessibility of the cellular landscape and its role in determining the diffusive routes adopted by molecules. The sensitivity of the pCF method toward detection of barriers means that different structural elements of the cell can be tested in terms of penetrability and mechanisms of regulation imparted on molecular flow. This has been recently demonstrated in a series of studies looking at molecular transport inside live cells. Here, we will review the theory behind detection of barriers to molecular flow, the rules to interpret pCF data, and relevant applications to intracellular transport.

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Near-Field Optical Study of Protein Transport Kinetics at a Single Nuclear Pore

Cited by 45 publications

References 44 publications

Self-regulated viscous channel in the nuclear pore complex

Self-regulated viscous channel in the nuclear pore complex

Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex

Measuring the flow of molecules in cells

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