Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3ß signalling.

ER-mitochondria contact sites (ERMCSs) regulate processes, including calcium homoeostasis, energy metabolism and autophagy. Previously, it was shown that during growth factor signalling, mTORC2/Akt gets recruited to and stabilizes ERMCSs. Independent studies showed that GSK3ß, a well-known Akt substrate, reduces ER-mitochondria connectivity by disrupting the VAPB-PTPIP51 tethering complex. However, the mechanisms that regulate ERMCSs are incompletely understood. Here we find that annulate lamellae (AL), relatively unexplored subdomains of ER enriched with a subset of nucleoporins, are present at ERMCSs. Depletion of Nup358, an AL-resident nucleoporin, results in enhanced mTORC2/Akt activation, GSK3ß inhibition and increased ERMCSs. Depletion of Rictor, a mTORC2-specific subunit, or exogenous expression of GSK3ß, was sufficient to reverse the ERMCS-phenotype in Nup358-deficient cells. We show that growth factor-mediated activation of mTORC2 requires the VAPB-PTPIP51 complex, whereas, Nup358's association with this tether restricts mTORC2/Akt signalling and ER-mitochondria connectivity. Expression of a Nup358 fragment that is sufficient for interaction with the VAPB-PTPIP51 complex suppresses mTORC2/Akt activation and disrupts ERMCSs. Collectively, our study uncovers a novel role for Nup358 in controlling ERMCSs by modulating the mTORC2/Akt/GSK3ß axis.

Spatial control of nucleoporin condensation by fragile X-related proteins.

Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation.

Importin α/ß and the tug of war to keep TDP-43 in solution: quo vadis?

The transactivation response-DNA binding protein of 43 kDa (TDP-43) is an aggregation-prone nucleic acid-binding protein linked to the etiology of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). These conditions feature the accumulation of insoluble TDP-43 aggregates in the neuronal cytoplasm that lead to cell death. The dynamics between cytoplasmic and nuclear TDP-43 are altered in the disease state where TDP-43 mislocalizes to the cytoplasm, disrupting Nuclear Pore Complexes (NPCs), and ultimately forming large fibrils stabilized by the C-terminal prion-like domain. Here, we review three emerging and poorly understood aspects of TDP-43 biology linked to its aggregation. First, how post-translational modifications in the proximity of TDP-43 N-terminal domain (NTD) promote aggregation. Second, how TDP-43 engages FG-nucleoporins in the NPC, disrupting the pore permeability and function. Third, how the importin α/ß heterodimer prevents TDP-43 aggregation, serving both as a nuclear import transporter and a cytoplasmic chaperone.

Interdependence between Nuclear Pore Gatekeepers and Genome Caretakers: Cues from Genome Instability Syndromes.

This review starts off with the first germline homozygous variants of the Nucleoporin 98 gene (NUP98) in siblings whose clinical presentation recalls Rothmund-Thomson (RTS) and Werner (WS) syndromes. The progeroid phenotype caused by a gene associated with haematological malignancies and neurodegenerative disorders primed the search for interplay between caretakers involved in genome instability syndromes and Nuclear Pore Complex (NPC) components. In the context of basic information on NPC architecture and functions, we discuss the studies on the interdependence of caretakers and gatekeepers in WS and Hereditary Fibrosing Poikiloderma (POIKTMP), both entering in differential diagnosis with RTS. In WS, the WRN/WRNIP complex interacts with nucleoporins of the Y-complex and NDC1 altering NPC architecture. In POIKTMP, the mutated FAM111B, recruited by the Y-complex's SEC13 and NUP96, interacts with several Nups safeguarding NPC structure. The linkage of both defective caretakers to the NPC highlights the attempt to activate a repair hub at the nuclear periphery to restore the DNA damage. The two separate WS and POIKTMP syndromes are drawn close by the interaction of their damage sensors with the NPC and by the shared hallmark of short fragile telomeres disclosing a major role of both caretakers in telomere maintenance.

Nucleoporin 50 mediates Kcna4 transcription to regulate cardiac electrical activity.

Emerging evidence has demonstrated that nucleoporins (Nups) play a pivotal role in cell-type-specific gene regulation, but how they control the expression and activity of ion channel genes in the heart remains unclear. Here, we show that Nup50, which is localized in the nucleus of cardiomyocytes, selectively induces an increase in the transcription and translation of Kcna4. The Kcna4 gene encodes a K+ voltage-gated channel of shaker-related subfamily member 4 and is essential for regulating the action potential in cardiac membranes. Using immunofluorescence imaging, luciferase assays and chromatin immunoprecipitation assays, we identified that the direct binding of the FG-repeat domain within Nup50 to the proximity of the Kcna4 promoter was required to activate the transcription and subsequent translation of Kcna4. Functionally, Nup50 overexpression increased the currents of KCNA4-encoded Ito,s channels, and reverse knockdown of Nup50 resulted in a remarkable decrease in the amplitude of Ito,s currents in cardiomyocytes. Moreover, a positive correlation between Nup50 and Kcna4 mRNA and protein expression was observed in heart tissues subjected to ischemic insults. These findings provide insights into the homeostatic control of cardiac electrophysiology through Nup-mediated regulation.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation.

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating expression of the HOXA gene locus during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5' HOXA genes, respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3' and 5' HOXA genes in the undifferentiated human NT2/D1 cell line. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. Whereas Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation. This article has an associated First Person interview with the first authors of the paper.

Interaction of nucleoporins with nuclear transport receptors: a structural perspective.

Soluble nuclear transport receptors and stationary nucleoporins are at the heart of the nucleocytoplasmic transport machinery. A subset of nucleoporins contains characteristic and repetitive FG (phenylalanine-glycine) motifs, which are the basis for the permeability barrier of the nuclear pore complex (NPC) that controls transport of macromolecules between the nucleus and the cytoplasm. FG-motifs can interact with each other and/or with transport receptors, mediating their translocation across the NPC. The molecular details of homotypic and heterotypic FG-interactions have been analyzed at the structural level. In this review, we focus on the interactions of nucleoporins with nuclear transport receptors. Besides the conventional FG-motifs as interaction spots, a thorough structural analysis led us to identify additional similar motifs at the binding interface between nucleoporins and transport receptors. A detailed analysis of all known human nucleoporins revealed a large number of such phenylalanine-containing motifs that are not buried in the predicted 3D-structure of the respective protein but constitute part of the solvent-accessible surface area. Only nucleoporins that are rich in conventional FG-repeats are also enriched for these motifs. This additional layer of potential low-affinity binding sites on nucleoporins for transport receptors may have a strong impact on the interaction of transport complexes with the nuclear pore and, thus, the efficiency of nucleocytoplasmic transport.

The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing.

Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing.

Identification of New FG-Repeat Nucleoporins with Amyloid Properties.

Amyloids are fibrillar protein aggregates with a cross-ß structure. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Functional amyloids with conservative amyloidogenic regions were found in different organisms. Protein aggregation appears to be beneficial for the organism in these cases. Therefore, this property might be conservative for orthologous proteins. The amyloid aggregates of the CPEB protein were suggested to play an important role in the long-term memory formation in Aplysia californica, Drosophila melanogaster, and Mus musculus. Moreover, the FXR1 protein demonstrates amyloid properties among the Vertebrates. A few nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human Nup153 and Nup58), are supposed or proved to form amyloid fibrils. In this study, we performed wide-scale bioinformatic analysis of nucleoporins with FG-repeats (phenylalanine-glycine repeats). We demonstrated that most of the barrier nucleoporins possess potential amyloidogenic properties. Furthermore, the aggregation-prone properties of several Nsp1 and Nup100 orthologs in bacteria and yeast cells were analyzed. Only two new nucleoporins, Drosophila melanogaster Nup98 and Schizosaccharomyces pombe Nup98, aggregated in different experiments. At the same time, Taeniopygia guttata Nup58 only formed amyloids in bacterial cells. These results rather contradict the hypothesis about the functional aggregation of nucleoporins.

[Assembly and Disassembly of the Nuclear Pore Complex: A View from the Structural Side].

Nucleocytoplasmic exchange in the cell occurs through the nuclear pore complexes (NPCs). NPCs are large multiprotein complexes with octagonal symmetry about their axis and imperfect mirror symmetry about a plane parallel with the nuclear envelop (NE). NPC fuses the inner and outer nuclear membranes and opens up a channel between nucleus and cytoplasm. NPC is built of nucleoporins. Each nucleoporin occurs in at least eight copies per NPC. Inside the NPC a permeability barrier forms by which NPCs can provide fast and selectable transport of molecules from one side of the nuclear membrane to the other. NPC architecture is based on hierarchical principle of organization. Nucleoporins are integrated into complexes that oligomerizes into bigger octomeric high-order structures. These structures are the main components of NPCs. In the first part of this work, the main attention is paid to NPC structure and nucleoporin properties. The second part is dedicated to mechanisms of NPC assembly and disassembly at different stages of the cell cycle.

Pathogenic Variants in NUP214 Cause "Plugged" Nuclear Pore Channels and Acute Febrile Encephalopathy.

We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as "plugs") in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.

Epigenetic regulation of neural stem cells: The emerging role of nucleoporins.

Nucleoporins (Nups) are components of the nuclear pore complex that, besides regulating nucleus-cytoplasmic transport, emerged as a hub for chromatin interaction and gene expression modulation. Specifically, Nups act in a dynamic manner both at specific gene level and in the topological organization of chromatin domains. As such, they play a fundamental role during development and determination of stemness/differentiation balance in stem cells. An increasing number of reports indicate the implication of Nups in many central nervous system functions with great impact on neurogenesis, neurophysiology, and neurological disorders. Nevertheless, the role of Nup-mediated epigenetic regulation in embryonic and adult neural stem cells (NSCs) is a field largely unexplored and the comprehension of their mechanisms of action is only beginning to be unveiled. After a brief overview of epigenetic mechanisms, we will present and discuss the emerging role of Nups as new effectors of neuroepigenetics and as dynamic platform for chromatin function with specific reference to the biology of NSCs.

Trafficking and/or division: Distinct roles of nucleoporins based on their location within the nuclear pore complex.

The nuclear pore complex (NPC) facilitates the trafficking of proteins and RNA between the nucleus and cytoplasm. The role of nucleoporins (Nups) in transport in the context of the NPC is well established, yet their function in tRNA export has not been fully explored. We selected several nucleoporins from different parts of the NPC to investigate their potential role in tRNA trafficking in Trypanosoma brucei. We show that while all of the nucleoporins studied are essential for cell viability, only TbNup62 and TbNup53a function in tRNA export. In contrast to homologs in yeast TbNup144 and TbNup158, which are part of the inner and outer ring of the NPC, have no role in nuclear tRNA trafficking. Instead, TbNup144 plays a critical role in nuclear division, highlighting the role of nucleoporins beyond nucleocytoplasmic transport. These results suggest that the location of nucleoporins within the NPC is crucial to maintaining various cellular processes.

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.

Into the basket and beyond: the journey of mRNA through the nuclear pore complex.

The genetic information encoded in nuclear mRNA destined to reach the cytoplasm requires the interaction of the mRNA molecule with the nuclear pore complex (NPC) for the process of mRNA export. Numerous proteins have important roles in the transport of mRNA out of the nucleus. The NPC embedded in the nuclear envelope is the port of exit for mRNA and is composed of ∼30 unique proteins, nucleoporins, forming the distinct structures of the nuclear basket, the pore channel and cytoplasmic filaments. Together, they serve as a rather stationary complex engaged in mRNA export, while a variety of soluble protein factors dynamically assemble on the mRNA and mediate the interactions of the mRNA with the NPC. mRNA export factors are recruited to and dissociate from the mRNA at the site of transcription on the gene, during the journey through the nucleoplasm and at the nuclear pore at the final stages of export. In this review, we present the current knowledge derived from biochemical, molecular, structural and imaging studies, to develop a high-resolution picture of the many events that culminate in the successful passage of the mRNA out of the nucleus.

Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy.

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

Identification and localization of Nup170 in the microsporidian Nosema bombycis.

As one of the core framework proteins of nuclear pore complex (NPC), nucleoporin Nupl70 acts as a structural adapter between the nucleolus and nuclear pore membrane and maintains the stability of NPC structure through interaction with other proteins. In this study, we identified a Nup170 protein in the microsporidian Nosema bombycis for the first time and named it as NbNup170. Secondary structure prediction showed that the NbNup170 contains α-helices and random coils. The three-dimensional structure of NbNup170 is elliptical in shape. Phylogenetic analysis based on the Nup170 and homologous sequences showed that N. bombycis clustered together with Vairimorpha ceranae and Vairimorpha apis. The immunofluorescence localization results showed that the NbNup170 was located on the plasma membrane of the dormant spore and transferred to the surface of sporoplasm in a punctate pattern when the dormant spore has finished germination, and that NbNup170 was distributed on the nuclear membrane and both sides of the nuclei of early proliferative phase, and only on the nuclear membrane during sporogonic phase in the N. bombycis. qPCR analysis showed that the relative expression level of NbNup170 maintained at a low level from 30 to 78 h post-infection with N. bombycis, then reached the highest at 102 h, while that of NbNup170 was repressed at a very low level throughout its life cycle by RNA interference. These results suggested that NbNup170 protein is involved in the proliferative phase and active during the sporogonic phase of N. bombycis.