Energetics of Transport through the Nuclear Pore Complex

Molecular transport across the nuclear envelope in eukaryotic cells is solely controlled by the nuclear pore complex (NPC). The NPC provides two types of nucleocytoplasmic transport: passive diffusion of small molecules and active chaperon-mediated translocation of large molecules. It has been shown that the interaction between intrinsically disordered proteins that line the central channel of the NPC and the transporting cargoes is the determining factor, but the exact mechanism of transport is yet unknown. Here, we use coarse-grained molecular dynamics simulations to quantify the energy barrier that has to be overcome for molecules to pass through the NPC. We focus on two aspects of transport. First, the passive transport of model cargo molecules with different sizes is studied and the size selectivity feature of the NPC is investigated. Our results show that the transport probability of cargoes is significantly reduced when they are larger than ∼5 nm in diameter. Secondly, we show that incorporating hydrophobic binding spots on the surface of the cargo effectively decreases the energy barrier of the pore. Finally, a simple transport model is proposed which characterizes the energy barrier of the NPC as a function of diameter and hydrophobicity of the transporting particles.     

Multiscale dynamics in nucleocytoplasmic transport

The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.     

Lighting up the nuclear pore complex

It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine-glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC.     

Brownian dynamics simulation of nucleocytoplasmic transport: a coarse-grained model for the functional state of the nuclear pore complex

The nuclear pore complex (NPC) regulates molecular traffic across the nuclear envelope (NE). Selective transport happens on the order of milliseconds and the length scale of tens of nanometers; however, the transport mechanism remains elusive. Central to the transport process is the hydrophobic interactions between karyopherins (kaps) and Phe-Gly (FG) repeat domains. Taking into account the polymeric nature of FG-repeats grafted on the elastic structure of the NPC, and the kap-FG hydrophobic affinity, we have established a coarse-grained model of the NPC structure that mimics nucleocytoplasmic transport. To establish a foundation for future works, the methodology and biophysical rationale behind the model is explained in details. The model predicts that the first-passage time of a 15 nm cargo-complex is about 2.6±0.13 ms with an inverse Gaussian distribution for statistically adequate number of independent Brownian dynamics simulations. Moreover, the cargo-complex is primarily attached to the channel wall where it interacts with the FG-layer as it passes through the central channel. The kap-FG hydrophobic interaction is highly dynamic and fast, which ensures an efficient translocation through the NPC. Further, almost all eight hydrophobic binding spots on kap-β are occupied simultaneously during transport. Finally, as opposed to intact NPCs, cytoplasmic filaments-deficient NPCs show a high degree of permeability to inert cargos, implying the defining role of cytoplasmic filaments in the selectivity barrier.     

Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane

Changes in nuclear pore complex (NPC) structure are studied following treatments modifying the cisternal calcium levels located between the two lipid bilayers that together form the nuclear envelope. Since the NPC forms the only known passageway across the nuclear envelope, it plays a central role in nucleocytoplasmic transport. Understanding the origin of conformational changes that may affect this trafficking or modify cargo interactions with the NPC is, therefore, necessary to completely understand the function of these complex molecules. In previous studies on the cytoplasmic side of the nuclear envelope, a central mass was observed in the pore of the NPC and its location was shown to be sensitive to the cisternal calcium levels. Here we report atomic force microscopy (AFM) measurements on the nuclear side of the envelope, which also reveal a cisternal calcium dependence in the conformational state of the NPC. These measurements, made at the single nuclear pore level, reveal a displacement of the central mass towards the nuclear side of the membrane following treatments with adenophostin A, a specific agonist of calcium channels (inositol 1,4,5-trisphosphate (IP(3)) receptors) located in the nuclear envelope. We further demonstrate that these conformational changes are observed in nuclear pores lacking the basket structure while samples prepared in the presence of protease inhibitors retain baskets and block AFM measurements of the channel. While these measurements are unable to distinguish whether the central mass is cargo or an integral component of the NPC, its dose-dependent displacement with cisternal calcium levels does suggest links to transport or to changes in cargo interactions with the NPC. Taken together with previous measurements done on the cytoplasmic side of the nuclear envelope, these studies argue against a piston-like displacement of the central mass and instead suggest a more complicated mechanism. One possibility involves a concerted collapse of the NPC rings towards one another following cisternal calcium release, thus leading to the apparent emergence of the central mass from each side of the NPC.     

A pathway separate from the central channel through the nuclear pore complex for inorganic ions and small macromolecules

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.     

Model Inspired by Nuclear Pore Complex Suggests Possible Roles for Nuclear Transport Receptors in Determining Its Structure

Nuclear transport receptors (NTRs) mediate nucleocytoplasmic transport via their affinity for unstructured proteins (polymers) in the nuclear pore complex (NPC). Here, we have modeled the effect of NTRs on polymeric structure in the nanopore confinement of the NPC central conduit. The model explicitly takes into account inter- and intramolecular interactions, as well as the finite size of the NTRs (∼20% of the NPC channel diameter). It reproduces various proposed scenarios for the channel structure, ranging from a central polymer condensate (selective phase) to brushlike polymer arrangements localized at the channel wall (virtual gate, reduction of dimensionality), with the transport receptors lining the polymer surface. In addition, it predicts a new structure in which NTRs become an integral part of the transport barrier by forming a cross-linked network with the unstructured proteins stretching across the pore. The model provides specific and distinctive predictions for the equilibrium spatial distributions of NTRs for these different scenarios that can be experimentally verified by, e.g., superresolution fluorescence microscopy. Moreover, it suggests mechanisms by which globular macromolecules (colloidal particles) can cause polymer-coated nanopores to switch between open and closed configurations, a possible explanation of the biological function of the NPC, and suggests potential technological applications for filtration and single-molecule sensing.     

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes.

In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.     

Correction

Nuclear transport receptors (NTRs) mediate nucleocytoplasmic transport via their affinity for unstructured proteins (polymers) in the nuclear pore complex (NPC). Here, we have modeled the effect of NTRs on polymeric structure in the nanopore confinement of the NPC central conduit. The model explicitly takes into account inter- and intramolecular interactions, as well as the finite size of the NTRs (∼20% of the NPC channel diameter). It reproduces various proposed scenarios for the channel structure, ranging from a central polymer condensate (selective phase) to brushlike polymer arrangements localized at the channel wall (virtual gate, reduction of dimensionality), with the transport receptors lining the polymer surface. In addition, it predicts a new structure in which NTRs become an integral part of the transport barrier by forming a cross-linked network with the unstructured proteins stretching across the pore. The model provides specific and distinctive predictions for the equilibrium spatial distributions of NTRs for these different scenarios that can be experimentally verified by, e.g., superresolution fluorescence microscopy. Moreover, it suggests mechanisms by which globular macromolecules (colloidal particles) can cause polymer-coated nanopores to switch between open and closed configurations, a possible explanation of the biological function of the NPC, and suggests potential technological applications for filtration and single-molecule sensing.     

Paradoxical relationships between active transport and global protein distributions in neurons

Neural function depends on continual synthesis and targeted trafficking of intracellular components, including ion channel proteins. Many kinds of ion channels are trafficked over long distances to specific cellular compartments. This raises the question of whether cargo is directed with high specificity during transit or whether cargo is distributed widely and sequestered at specific sites. We addressed this question by experimentally measuring transport and expression densities of Kv4.2, a voltage-gated transient potassium channel that exhibits a specific dendritic expression that increases with distance from the soma and little or no functional expression in axons. In over 500 h of quantitative live imaging, we found substantially higher densities of actively transported Kv4.2 subunits in axons as opposed to dendrites. This paradoxical relationship between functional expression and traffic density supports a model—commonly known as the sushi belt model—in which trafficking specificity is relatively low and active sequestration occurs in compartments where cargo is expressed. In further support of this model, we find that kinetics of active transport differs qualitatively between axons and dendrites, with axons exhibiting strong superdiffusivity, whereas dendritic transport resembles a weakly directed random walk, promoting mixing and opportunity for sequestration. Finally, we use our data to constrain a compartmental reaction-diffusion model that can recapitulate the known Kv4.2 density profile. Together, our results show how nontrivial expression patterns can be maintained over long distances with a relatively simple trafficking mechanism and how the hallmarks of a global trafficking mechanism can be revealed in the kinetics and density of cargo.     

The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion

Nuclear pore complexes (NPCs) restrict the nucleocytoplasmic flux of most macromolecules, but permit facilitated passage of nuclear transport receptors and their cargo complexes. We found that a simple hydrophobic interaction column can mimic the selectivity of NPCs surprisingly well and that nuclear transport receptors appear to be the most hydrophobic soluble proteins. This suggests that surface hydrophobicity represents a major sorting criterion of NPCs. The rate of NPC passage of cargo-receptor complexes is, however, not dominated just by properties of the receptors. We found that large cargo domains drastically hinder NPC passage and require more than one receptor molecule for rapid translocation. This argues against a rigid translocation channel and instead suggests that NPC passage involves a partitioning of the entire translocating species into a hydrophobic phase, whereby the receptor:cargo ratio determines the solubility in that permeability barrier. Finally, we show that interfering with hydrophobic interactions causes a reversible collapse of the permeability barrier of NPCs, which is consistent with the assumption that the barrier is formed by phenylalanine-rich nucleoporin repeats that attract each other through hydrophobic interactions.     

Exceptional structural and mechanical flexibility of the nuclear pore complex

Nuclear pore complexes (NPCs) mediate all transport between the cytosol and the nucleus and therefore take centre stage in physiology. While transport through NPCs has been extensively investigated little is known about their structural and barley anything about their mechanical flexibility. Structural and mechanical flexibility of NPCs, however, are presumably of key importance. Like the cell and the cell nucleus, NPCs themselves are regularly exposed to physiological mechanical forces. Besides, NPCs reveal striking transport properties which are likely to require fairly high structural flexibility. The NPC transports up to 1,000 molecules per second through a physically 9 nm wide channel which repeatedly opens to accommodate macromolecules significantly larger than its physical diameter. We hypothesised that NPCs possess remarkable structural and mechanical stability. Here, we tested this hypothesis at the single NPC level using the nano‐imaging and probing approach atomic force microscopy (AFM). AFM presents the NPC as a highly flexible structure. The NPC channel dilates by striking 35% on exposure to trans‐cyclohexane‐1,2‐diol (TCHD), which is known to transiently collapse the hydrophobic phase in the NPC channel like receptor–cargo complexes do in transit. It constricts again to its initial size after TCHD removal. AFM‐based nano‐indentation measurements show that the 50 nm long NPC basket can astonishingly be squeezed completely into the NPC channel on exposure to incremental mechanical loads but recovers its original vertical position within the nuclear envelope plane when relieved. We conclude that the NPC possesses exceptional structural and mechanical flexibility which is important to fulfilling its functions. J. Cell. Physiol. 226: 675–682, 2011. © 2010 Wiley‐Liss, Inc.     

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.     

An agent-based model for mRNA export through the nuclear pore complex

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

Nucleoporin's Like Charge Regions Are Major Regulators of FG Coverage and Dynamics Inside the Nuclear Pore Complex

Nucleocytoplasmic transport has been the subject of a large body of research in the past few decades. Recently, the focus of investigations in this field has shifted from studies of the overall function of the nuclear pore complex (NPC) to the examination of the role of different domains of phenylalanine-glycine nucleoporin (FG Nup) sequences on the NPC function. In our recent bioinformatics study, we showed that FG Nups have some evolutionarily conserved sequence-based features that might govern their physical behavior inside the NPC. We proposed the ‘like charge regions’ (LCRs), sequences of charged residues with only one type of charge, as one of the features that play a significant role in the formation of FG network inside the central channel. In this study, we further explore the role of LCRs in the distribution of FG Nups, using a recently developed coarse-grained molecular dynamics model. Our results demonstrate how LCRs affect the formation of two transport pathways. While some FG Nups locate their FG network at the center of the NPC forming a homogeneous meshwork of FG repeats, other FG Nups cover the space adjacent to the NPC wall. LCRs in the former group, i.e. FG Nups that form an FG domain at the center, tend to regulate the size of the highly dense, doughnut-shaped FG meshwork and leave a small low FG density area at the center of the pore for passive diffusion. On the other hand, LCRs in the latter group of FG Nups enable them to maximize their interactions and cover a larger space inside the NPC to increase its capability to transport numerous cargos at the same time. Finally, a new viewpoint is proposed that reconciles different models for the nuclear pore selective barrier function.     

Conserved Spatial Organization of FG Domains in the Nuclear Pore Complex

Selective transport through the nuclear pore complex (NPC) requires nucleoporins containing natively unfolded phenylalanine-glycine (FG) domains. Several differing models for their dynamics within the pore have been proposed. We characterize the behavior of the FG nucleoporins in vivo using polarized fluorescence microscopy. Using nucleoporins tagged with green fluorescent protein along their FG domains, we show that some of these proteins are ordered, indicating an overall orientational organization within the NPC. This orientational ordering of the FG domains depends on their specific context within the NPC, but is independent of active transport and cargo load. For most nups, behavior does not depend on the FG motifs. These data support a model whereby local geometry constrains the orientational organization of the FG nups. Intriguingly, homologous yeast and mammalian proteins show conserved behavior, suggesting functional relevance. Our findings have implications for mechanistic models of NPC transport.