Nuclear Pore Complex Permeability Research Articles

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.

Understanding the role of mechanics in nucleocytoplasmic transport.

Cell nuclei are submitted to mechanical forces, which in turn affect nuclear and cell functions. Recent evidence shows that a crucial mechanically regulated nuclear function is nucleocytoplasmic transport, mediated by nuclear pore complexes (NPCs). Mechanical regulation occurs at two levels: first, by force application to the nucleus, which increases NPC permeability likely through NPC stretch. Second, by the mechanical properties of the transported proteins themselves, as mechanically labile proteins translocate through NPCs faster than mechanically stiff ones. In this perspective, we discuss this evidence and the associated mechanisms by which mechanics can regulate the nucleo-cytoplasmic partitioning of proteins. Finally, we analyze how mechanical regulation of nucleocytoplasmic transport can provide a systematic approach to the study of mechanobiology and open new avenues both in fundamental and applied research.

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.

Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

Gemcitabine and Thapsigargin Counteract the Effects of 5‐Fluorouracil on Nuclear Transport during Apoptosis through Different Mechanisms of Action

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are disrupted and dismantled. Specifically, 5‐flurouracil (5‐FU) increases NPC permeability and disrupts the Ran gradient, decreasing it's nuclear levels and inhibiting nuclear export. As a result, combination of 5‐FU with other chemotherapeutics, such as topotican, increases their efficiency, by retaining pro‐apoptotic factors in the nucleus. Here we examine the ability of 5‐FU to enhance the effects of another drug, gemcitabine, previously shown to work synergistically with nuclear transport inhibitors. Interestingly, we found that while both 5‐FU and gemcitabine cause a decrease in nuclear Ran, combination of the drugs led to no significant change in Ran localization. In order to determine the mechanism of action for both drugs and the means of their counteraction, we explored their effects on NPC permeability. As expected, 5‐FU increased NPC permeability; however, gemcitabine did not disrupt the NPC. Additionally, gemcitabine counteracted 5‐FU, indicating that their antagonistic effects on Ran localization occur at the level of NPC permeability. As 5‐FU's effects on the NPC are known to be calcium dependent, we also explored whether gemcitabine similarly counteracts thapsigargin, an inhibitor of ER calcium uptake and known disruptor of nuclear Ran localization. Interestingly, gemcitabine had no effect on thapsigargin's ability to decrease nuclear Ran levels. Furthermore, thapsigargin had no effect on NPC permeability. Instead, thapsigargin counteracted the ability of 5‐FU to decrease nuclear Ran levels, despite the fact that it was unable to change 5‐FU induced NPC permeability. These results suggest that gemcitabine and thapsigargin mediate similar negative effects on Ran and nuclear transport counteractive to 5‐FU, but thapsigargin does so via an unknown mechanism. Therefore, the effects of 5‐FU and thapsigargin may reflect various potential means of calcium mediated changes to nuclear transport in early apoptosis. Future work will involve exploring the direct impact of these treatments on calcium levels. Importantly, the impacts of all drug treatments on Ran in the nucleus were due to changes in cellular localization differences alone, as immunoblot analysis demonstrated no changes in Ran protein expression levels. The combinatory effects of these drugs on the NPC and nuclear transport were unexpected based on previous work, and indicate the wide variation in nuclear disruption in apoptosis. At this level it is unclear if the impacts of 5‐FU, thapsigargin, and gemcitabine on nuclear transport and the NPC alone will be sufficient to efficiently predict the efficacy of these combinations on apoptosis. The counteraction of gemcitabine or thapsigargin on 5‐FU at this level may or may not be a predictor of cell death, and thus our ongoing work involves studies of cell viability and the localization of apoptotic factors with these treatments. Understanding the molecular mechanisms of these drugs will help to inform the usefulness of their combination in cancer treatment, particularly in cases of drug resistance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

5-Flurouracil disrupts nuclear export and nuclear pore permeability in a calcium dependent manner.

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are slowly disrupted and dismantled. 5-Fluorouracil (5-FU) and the camptothecin derivatives irinotecan and topotecan, are linked to altered nuclear transport of specific proteins; however, their general effects on the NPC and transport during apoptosis have not been characterized. We demonstrate that 5-FU, but not topotecan, increases NPC permeability, and disrupts Ran-mediated nuclear transport before the disruption of the NPC. This increased permeability is dependent on increased cellular calcium, as the Ca2+ chelator BAPTA-AM, abolishes the effect. Furthermore, increased calcium alone was sufficient to disrupt the Ran gradient. Combination treatments of 5-FU with topotecan or irinotecan, similarly disrupted nuclear transport before disassembly of the NPC. In both single and combination treatments nuclear transport was disrupted before caspase 9 activation, indicating that 5-FU induces an early caspase-independent increase in NPC permeability and alteration of nuclear transport. Because Crm1-mediated nuclear export of tumor suppressors is linked to drug resistance we also examined the effect of 5-FU on the nuclear export of a specific target, topoisomerase. 5-FU treatment led to accumulation of topoisomerase in the nucleus and recovered the loss nuclear topoisomerase induced by irinotecan or topotecan, a known cause of drug resistance. Furthermore, 5-FU retains its ability to cause nuclear accumulation of p53 in the presence of irinotecan or topotecan. Our results reveal a new mechanism of action for these therapeutics during apoptosis, opening the door to other potential combination chemotherapies that employ 5-FU as a calcium mediated inhibitor of Crm1-induced nuclear export of tumor suppressors.

O-GlcNAc-ylation in the Nuclear Pore Complex.

O-GlcNAc-ylation is the post-translational addition of an O-linked β-N-acetylglucosamine to the serine and threonine residues of thousands of proteins in eukaryotic cells. Specifically, half of the thirty different types of protein components in the nuclear pore complex (NPC) are modified by O-GlcNAc, of which the majority are intrinsically disordered nucleoporins (Nups) containing multiple phenylalanine-glycine (FG) repeats. Moreover, these FG-Nups form a strict selectivity barrier with a high density of O-GlcNAc in the NPC to mediate bidirectional trafficking between the cytoplasm and nucleus. However, the roles that O-GlcNAc plays in the structure and function of the NPC remain obscure. In this review paper, we will discuss the current knowledge of O-GlcNAc-ylated Nups, highlight some new techniques used to probe O-GlcNAc's roles in the nuclear pore, and finally propose a new model for the effect of O-GlcNAc on the NPC's permeability.

Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A(pro).

Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection, leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport, the mechanisms and consequences of Nup98 proteolysis were examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2A(pro)). Incubation of bacterially expressed or in vitro-translated Nup98 with 2A(pro) results in proteolytic cleavage at multiple sites in vitro, indicating that 2A(pro) cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2A(pro) proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains the overall NPC structure and leaves other transport pathways unaffected. Enteroviruses are dependent upon host nuclear RNA binding proteins for efficient replication. This study examines the mechanisms responsible for alterations in nuclear transport in enterovirus-infected cells that lead to the cytoplasmic accumulation of these proteins. The results demonstrate that the enterovirus 2A protease directly cleaves the nuclear pore complex (NPC) protein, Nup98, at amino acid positions G374 and G552 both in vitro and in infected cells. Cleavage at these positions results in the selective removal of the FG-containing N terminus of Nup98 from the NPC, while the C terminus remains associated. Nup88, a core component of the NPC that serves as a docking site for the C terminus of Nup98, remains associated with the NPC in infected cells. These findings help to explain the alterations in permeability and nuclear transport in enterovirus-infected cells and how NPCs remain functional for certain trafficking pathways despite significant alterations to their compositions.

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.

Altering nuclear pore complex function impacts longevity and mitochondrial function in S. cerevisiae.

The eukaryotic nuclear permeability barrier and selective nucleocytoplasmic transport are maintained by nuclear pore complexes (NPCs), large structures composed of ∼ 30 proteins (nucleoporins [Nups]). NPC structure and function are disrupted in aged nondividing metazoan cells, although it is unclear whether these changes are a cause or consequence of aging. Using the replicative life span (RLS) of Saccharomyces cerevisiae as a model, we find that specific Nups and transport events regulate longevity independent of changes in NPC permeability. Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100-null mutants. We show that Nup116 mediates nuclear import of the karyopherin Kap121, and each protein is required for mitochondrial function. Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast. Overexpression of GSP1, the small GTPase that powers karyopherin-mediated transport, rescued mitochondrial and RLS defects in nup116 mutants and increased longevity in wild-type cells. Together, these studies reveal that specific NPC nuclear transport events directly influence aging.

Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner.

Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.

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.

Alterations In Binding Properties Of Myocardial Nuclear Membrane Receptors Induce Nuclear Calcium Overload In Rat Ischemia-reperfusion

Cell nuclei possess an independent calcium regulatory system consisting of nuclear Ca2+-ATPases (NCA), IP3 receptors (IP3R), IP4 receptors (IP4R), ryanodine receptors(RyR), and nuclear pore complexes(NPC). We studied the changes in Ca2+n and its regulatory system in rat model of myocardial ischemia-reperfusion injury(IRI) induced by 30 min coronary occlusion followed by 180 min reperfusion. The Ca2+n content in isolated nuclei was measured with atomic absorption spectrophotometer. NPC permeability was assessed through the amount of calmodulin conjugated Alexa FluoTM 488 as fluorescent probes. NCA activity was evaluated by phosphate group released from ATP in enzymatic reaction. The maximum binding capacity(Bmax) and dissociation constant(Kd) of IP3R, IP4R and RyR were determined by radioligand binding of [3H]IP4, [3H]IP4 and [3H]-ryanodine to isolated cardiomyocytic nuclei. All results are shown in the table. Our findings suggest that in vivo rat myocardial IRI is characterized by Ca2+n overload, upregulations of nuclear IP3R and IP4R, downregulations of NCA activity and RyR, and an increase in permeability of NPC. The upregulation of nuclear IP3R and IP4R may be responsible for the nuclear calcium overload.

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.

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.

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.