Permeability Barrier Of Nuclear Pore Complexes Research Articles

The in-cell liquid-like state of the nuclear pore complex permeability barrier

The in-cell liquid-like state of the nuclear pore complex permeability barrier

SUN1 facilitates CHMP7 nuclear influx and injury cascades in sporadic amyotrophic lateral sclerosis.

We have recently identified the aberrant nuclear accumulation of the ESCRT-III protein CHMP7 as an initiating event that leads to a significant injury to the nuclear pore complex (NPC) characterized by the reduction of specific nucleoporins from the neuronal NPC in sporadic amyotrophic lateral sclerosis (sALS) and C9orf72 ALS/frontotemporal dementia (FTD)-induced pluripotent stem cell-derived neurons (iPSNs), a phenomenon also observed in post-mortem patient tissues. Importantly, this NPC injury is sufficient to contribute to TDP-43 dysfunction and mislocalization, a common pathological hallmark of neurodegenerative diseases. However, the molecular mechanisms and events that give rise to increased nuclear translocation and/or retention of CHMP7 to initiate this pathophysiological cascade remain largely unknown. Here, using an iPSN model of sALS, we demonstrate that impaired NPC permeability barrier integrity and interactions with the LINC complex protein SUN1 facilitate CHMP7 nuclear localization and the subsequent 'activation' of NPC injury cascades. Collectively, our data provide mechanistic insights in the pathophysiological underpinnings of ALS/FTD and highlight SUN1 as a potent contributor to and modifier of CHMP7-mediated toxicity in sALS pathogenesis.

A survey of the specificity and mechanism of 1,6 hexanediol-induced disruption of nuclear transport

ABSTRACT Selective transport through the nuclear pore complex (NPC) depends on the dynamic binding of FG-repeat containing nucleoporins, the FG-nups, with each other and with Karyopherins (Kaps). Here, we assessed the specificity and mechanism by which the aliphatic alcohol 1,6-hexanediol (1,6HD) disrupts the permeability barrier of NPCs in live baker’s yeast cells. After a 10-minute exposure to 5% 1,6HD, no notable changes were observed in cell growth, cytosolic pH and ATP levels, or the appearance of organelles. However, effects on the cytoskeleton and Hsp104 were noted. 1,6HD clearly affected the NPC permeability barrier, allowing passive nuclear entry of a 177kDa reporter protein that is normally confined to the cytosol. Moreover, multiple Kaps were displaced from NPCs, and the displacement of Kap122-GFP correlated with the observed passive permeability changes. 1,6HD thus temporarily permeates NPCs, and in line with Kap-centric models, the mechanism includes the release of numerous Kaps from the NPCs.

On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions.

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the β-karyopherin (Kapβ) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapβs, cargoes and Ran to establish the asymmetry of NCT.

Following a Giant's Footsteps: Single-Particle and Super-Resolution Approaches to Decipher the Nuclear Transport of Hepatitis B Virus Capsids

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), and is a tightly regulated process. The Hepatitis B Virus (HBV) capsid is one of the largest cargoes ever shown to be imported intact to the nuclear side of the NPC. The import of such large cargoes - close in dimension to the expected maximum size of the NPC central channel - is largely unknown, as well as to what extent plasticity of the NPC transport conduit is required to accommodate the transport. To decipher the molecular details of this host-pathogen interaction, we developed a dual strategy that allows us to visualize with high spatiotemporal resolution the viral capsid, as well as the elusive permeability barrier of the NPC. We follow nuclear import of fluorescently labelled HBV capsids in mammalian cells using single-particle tracking on a purpose optimized self-designed HILO microscope. We observe an average transport time in the hundreds of milliseconds range, orders of magnitude faster than what expected for such a large cargo. In addition, slower interactions occur at the cytoplasmic filaments and nuclear basket, pointing to docking and undocking interactions being the rate limiting factor. Complementarily, to decipher the changes occurring in the NPC during viral transport, we developed a method to image site-specifically labelled nucleoporins in the cell using super-resolution microscopy (SRM). We combine site-specific tagging of proteins, through an enhanced Amber suppression system, with DNA-PAINT microscopy, enabling the visualization of even low abundance proteins such as nucleoporins. This synergistic approach gives us the potential to resolve the plasticity of the permeability barrier of NPCs, allowing us a dual view on the viral transport from both the cargo and the “gate” (NPC) level in an unprecedented manner.

Nucleoporin-Regulated MAP Kinase Signaling in Immunity to a Necrotrophic Fungal Pathogen.

Pathogen-responsive mitogen-activated protein kinase (MAPK or MPK) cascades relay signals from activated immune receptors across the nuclear envelope to intranuclear targets. However, in plants, little is known about the spatial control of MAPK signaling. Here, we report that the Arabidopsis (Arabidopsis thaliana) nuclear pore complex protein Nup88/MOS7 is essential for immunity to the necrotrophic fungus Botrytis cinerea The mos7-1 mutation, causing a four-amino acid deletion, compromises B. cinerea-induced activation of the key immunoregulatory MAPKs MPK3/MPK6 and reduces MPK3 protein levels posttranscriptionally. Furthermore, MOS7 contributes to retaining a sufficient MPK3 abundance in the nucleus, which is required for full immunity to B. cinerea Finally, we present a structural model of MOS7 and show that the mos7-1 mutation compromises interactions with Nup98a/b, two phenylalanine-glycine repeat nucleoporins implicated in maintaining the selective nuclear pore complex permeability barrier. Together, our analysis uncovered MOS7 and Nup98 as novel components of plant immunity toward a necrotrophic pathogen and provides mechanistic insights into how these nucleoporins coordinate nucleocytoplasmic transport to mount a robust immune response.

Clathrin inhibitor Pitstop-2 disrupts the nuclear pore complex permeability barrier.

Existence of a selective nucleocytoplasmic permeability barrier is attributed to Phenylalanine-Glycine rich proteins (FG-nups) within the central channel of the nuclear pore complex (NPC). Limited understanding of the FG-nup structural arrangement hinders development of strategies directed at disrupting the NPC permeability barrier. In this report we explore an alternative approach to enhancing the NPC permeability for exogenous macromolecules. We demonstrate that the recently discovered inhibitor of clathrin coat assembly Pitstop-2 compromises the NPC permeability barrier in a rapid and effective manner. Treatment with Pitstop-2 causes a collapse of the NPC permeability barrier and a reduction of Importin β binding accompanied by alteration of the NPC ultrastructure. Interestingly, the effects are induced by the same chemical agent that is capable of inhibiting clathrin-mediated endocytosis. To our knowledge, this is the first functional indication of the previously postulated evolutionary relation between clathrin and NPC scaffold proteins.

Viruses Challenge Selectivity Barrier of Nuclear Pores

Exchange between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs) embedded in the double membrane of the nuclear envelope. NPC permeability barrier restricts the entry of inert molecules larger than 5 nm in diameter but allows facilitated entry of selected cargos, whose size can reach up to 39 nm. The translocation of large molecules is facilitated by nuclear transport receptors (NTRs) that have affinity to proteins of NPC permeability barrier. Viruses that enter the nucleus replicate evolved strategies to overcome this barrier. In this review, we will discuss the functional principles of NPC barrier and nuclear transport machinery, as well as the various strategies viruses use to cross the selective barrier of NPCs.

The Supramolecular Assembly of Intrinsically Disordered Nucleoporin Domains is Tuned by Inter-Chain Interactions

Nuclear pore complexes (NPCs) are the gates that mediate the exchange of all macromolecules between the cytoplasm and the nucleus. They are highly selective: inert proteins bigger than 30 kDa cannot diffuse through NPCs unless they are bound to nuclear transport receptors (NTRs). This selectivity arises from a supramolecular assembly of intrinsically unfolded nucleoporin domains which contain phenylalanine-glycine (FG)-rich repeats, so called FG-repeat domains (FGRDs). Different models for the size and species selectivity of the permeability barrier have been suggested that build on different putative supramolecular assemblies of FGRDs. A controversially discussed question today is whether and how inter-FGRD interactions contribute to the function of the permeability barrier. We have studied the inter-FGRD interactions in monolayers of end-grafted FGRDs which constitute a biomimetic nano-scale model system of the NPC's permeability barrier. A toolbox of biophysical characterization techniques enabled us to relate the strength of inter-FGRD interactions of different FGRD types to monolayer formation and morphology. Our main findings are that inter-FGRD interactions strongly affect the kinetics of formation, the morphology (thickness and lateral homogeneity), and the mechanical properties of FGRD monolayers. Based on these results, we propose that the strength of inter-FGRD interactions in the NPC has evolved to promote the formation of a homogeneous meshwork with a small mesh size. Our results highlight the importance of inter-FGRD interactions for the functionality, and hence contribute important information to refine the model of transport across the nuclear pore permeability barrier.

Importin-Beta and Ran Regulate the Passive Permeability Barrier in the Nuclear Pore Complex

The nuclear pore complex (NPC) is a large, multi-protein assembly that mediates the selective transport of molecules between the cytoplasm and nucleus in eukaryotic cells. While the proteins involved in the transport pathway have largely been identified, the physical mechanism by which this complex can support both efficient and selective molecular transport remains unclear. Using fluorescence microscopy and super-resolution imaging techniques, we have examined how nuclear transport receptors influence the permeability properties of the NPC for both active and passive transport processes. We find that importin-β binding to the nucleoporin Nup153 significantly slows passive transport through the NPC; however, Ran in its GTP-bound form reverses this effect. FRAP studies reveal that RanGTP weakens the binding interaction of importin-β to the NPC. Furthermore, STORM imaging of individual importin-β localizations inside the NPC show that RanGTP dramatically changes the importin-β distribution within the channel. These results suggest that importin-β in conjunction with Nup153 is an integral component of the NPC permeability barrier which is regulated by RanGTP.

Structural organization of the nuclear pore permeability barrier

The efficiency of gene therapy in non-dividing cells is particularly poor due to restricted nuclear delivery rates of exogenously applied macromolecules across the nuclear pore complexes (NPCs). Therefore, improved intranuclear delivery of transgenes requires an ability to modulate the barrier function of the NPC. Despite a large body of experimental evidence accumulated to date, the contribution of individual NPC proteins (nucleoporins) to the formation of the NPC permeability barrier as well as their structural organization within the NPC remains under debate. In the present study, we revisit the view on the spatial arrangement of the Phe-Gly rich domains (FG-domains) of a subset of nucleoporins known as FG-nucleoporins. They are generally believed to be the key constituents of the NPC permeability barrier. Comparison of the binding pattern of a transport receptor importin β fragment, that binds specifically to FG-domains, with the binding pattern of wheat germ agglutinin that binds elsewhere in the NPC, reveals that FG-domains tend to cluster in the very center of the NPC. Furthermore, a controlled sequential release of the barrier-forming nucleoporins results in a gradual breakdown of the NPC permeability barrier. The breakdown is initiated by a dissociation of Nup62 from the NPC. This is accompanied by an increased passive diffusion of small molecules across the NPC. Subsequent dissociation of Nup98 and possibly other nucleoporins results in a collapse of the barrier for larger molecules. We therefore conclude that FG-nucleoporins do not contribute equally to the maintenance of the NPC permeability barrier exclusion limit. This implies that a controlled release of nucleoporins that contribute most to the formation and maintenance of the NPC barrier can facilitate access of therapeutic macromolecules into the nucleus.

Domain topology of nucleoporin Nup98 within the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.

Characterisation of the passive permeability barrier of nuclear pore complexes

Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve-like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross-linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant-negative importin β45-462 fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC-passing molecule. They have little effect on small species, inhibit the passage of green fluorescent protein-sized objects >10-fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties

The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo-cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than ≈30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation-promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repeat type, can also form highly selective hydrogels. While supporting massive, reversible importin-mediated cargo influx, FG/FxFG, GLFG or mixed hydrogels remained firm barriers towards inert objects that lacked nuclear transport signals. This indicates that FG hydrogels immediately reseal behind a translocating species and thus possess ‘self-healing' properties. NTRs not only left the barrier intact, they even tightened it against passive influx, pointing to a role for NTRs in establishing and maintaining the permeability barrier of NPCs.

Intramolecular Cohesion of Coils Mediated by Phenylalanine–Glycine Motifs in the Natively Unfolded Domain of a Nucleoporin

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine–glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin–cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

Natively Unfolded Nucleoporins Gate Protein Diffusion across the Nuclear Pore Complex

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.

Scientists Share Nuclear Secrets at Jekyll Island

Scientists Share Nuclear Secrets at Jekyll Island

The strategy for coupling the RanGTP gradient to nuclear protein export.

The Ran GTPase plays critical roles in both providing energy for and determining the directionality of nucleocytoplasmic transport. The mechanism that couples the RanGTP gradient to nuclear protein export will determine the rate of and limits to accumulation of export cargoes in the cytoplasm, but is presently unknown. We reasoned that plausible coupling mechanisms could be distinguished by comparing the rates of reverse motion of export cargoes through the nuclear pore complex (NPC) with the predictions of a mathematical model. Measurement of reverse export rates in Xenopus oocytes revealed that nuclear export signals can facilitate RanGTP-dependent cargo movement into the nucleus against the RanGTP gradient at rates comparable to export rates. Although export cargoes with high affinity for their receptor are exported faster than those with low affinity, their reverse transport is also greater. The ratio of the rates of reverse and forward export of a cargo is proportional to its rate of diffusion through the NPC, i.e., to the ability of the cargo to penetrate the NPC permeability barrier. The data substantiate a diffusional mechanism of coupling and suggest the existence of a high concentration of RanGTP-receptor complexes within the NPC that decreases sharply at the cytoplasmic boundary of the NPC permeability barrier.

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.