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The nuclear pore complex (NPC) serves as a pivotal subcellular structure, acting as a gateway that orchestrates nucleocytoplasmic transport through a selectively permeable barrier. Nucleoporins (Nups), particularly those containing phenylalanine-glycine (FG) motifs, play indispensable roles within this barrier. Recent advancements in technology have significantly deepened our understanding of the NPC's architecture and operational intricacies, owing to comprehensive investigations. Nevertheless, the conspicuous presence of intrinsically disordered regions within FG-Nups continues to present a formidable challenge to conventional static characterization techniques. Historically, a multitude of strategies have been employed to unravel the intricate organization and behavior of FG-Nups within the NPC. These endeavors have given rise to multiple models that strive to elucidate the structural layout and functional significance of FG-Nups. Within this exhaustive review, we present a comprehensive overview of these prominent models, underscoring their proposed dynamic and structural attributes, supported by pertinent research. Through a comparative analysis, we endeavor to shed light on the distinct characteristics and contributions inherent in each model. Simultaneously, it remains crucial to acknowledge the scarcity of unequivocal validation for any of these models, as substantiated by empirical evidence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rush, Jiang, Tingey, Feng and Yang.)

The nuclear pore complex (NPC) is among the most elaborate protein complexes in eukaryotes. While ribosomes and proteasomes are known to require dedicated assembly machinery, our understanding of NPC assembly is at a relatively early stage. Defects in NPC assembly or homeostasis are tied to movement disorders, including dystonia and amyotrophic lateral sclerosis (ALS), as well as aging, requiring a better understanding of these processes to enable therapeutic intervention. Here, we discuss recent progress in the understanding of NPC assembly and highlight how related defects in human disorders can shed light on NPC biogenesis. We propose that the condensation of phenylalanine-glycine repeat nucleoporins needs to be carefully controlled during NPC assembly to prevent aberrant condensation, aggregation, or amyloid formation., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A journey from the earliest known use of lenses and magnifying glasses in ancient times, through the development of microscopes and towards modern electron microscopy techniques. The evolving technology and improved microscopes enabled the discovery of intracellular organelles, the nucleus and nuclear pore complexes (NPCs). Current advances have led to composite three-dimensional models showing NPC structure in unprecedented detail but relying on the averaging of many images. A complementary approach is field emission scanning electron microscopy providing topographic surface images that are easily and intuitively interpreted by our brain. Recent advances in this technique have made it possible to expose nuclei from human cells and to focus on individual NPCs and their architectural features., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

One of the remarkable features of eukaryotes is the nucleus, delimited by the nuclear envelope (NE), a complex structure and home to the nuclear lamina and nuclear pore complex (NPC). For decades, these structures were believed to be mainly architectural elements and, in the case of the NPC, simply facilitating nucleocytoplasmic trafficking. More recently, the critical roles of the lamina, NPC and other NE constituents in genome organisation, maintaining chromosomal domains and regulating gene expression have been recognised. Importantly, mutations in genes encoding lamina and NPC components lead to pathogenesis in humans, while pathogenic protozoa disrupt the progression of normal development and expression of pathogenesis-related genes. Here, we review features of the lamina and NPC across eukaryotes and discuss how these elements are structured in trypanosomes, protozoa of high medical and veterinary importance, highlighting lineage-specific and conserved aspects of nuclear organisation., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Nuclear pore complexes (NPCs) play a critical role in maintaining the equilibrium between the nucleus and cytoplasm, enabling bidirectional transport across the nuclear envelope, and are essential for proper nuclear organization and gene regulation. Perturbations in the regulatory mechanisms governing NPCs and nuclear envelope homeostasis have been implicated in the pathogenesis of several neurodegenerative diseases. The ESCRT-III pathway emerges as a critical player in the surveillance and preservation of well-assembled, functional NPCs, as well as nuclear envelope sealing. Recent studies have provided insights into the involvement of nuclear ESCRT-III in the selective reduction of specific nucleoporins associated with neurodegenerative pathologies. Thus, maintaining quality control of the nuclear envelope and NPCs represents a pivotal element in the pathological cascade leading to neurodegenerative diseases. This review describes the constituents of the nuclear-cytoplasmic transport machinery, encompassing the nuclear envelope, NPC, and ESCRT proteins, and how their structural and functional alterations contribute to the development of neurodegenerative diseases., (© 2023 Federation of European Biochemical Societies.)

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinβ1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.

The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super‐resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically‐encoded self‐labeling enzymes such as SNAP‐tag and HaloTag with DNA‐PAINT microscopy. We resolve single copies of nucleoporins in the human Y‐complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy. [ABSTRACT FROM AUTHOR]

During my postdoc interview in June of 1998, I asked Günter why he was moving more towards the nucleus in his latest studies. He said, "Well Joe, that's where everything starts." By the end of the interview, I accepted the postdoc. He had a way of making everything sound so cool. Günter's progression was natural, since the endoplasmic reticulum and the nucleus are the only organelles that share the same membrane. The nuclear envelope extends into a double membrane system with nuclear pore complexes embedded in the pore membrane openings. Even while writing this review, I remember Günter stressing; it is the nuclear pore complex. Just saying nuclear pore doesn't encompass the full magnitude of its significance. The nuclear pore complex is one of the largest collection of proteins that fit together for an overall function: transport. This review will cover the Blobel lab contributions in the quest for the blueprint of the nuclear pore complex from isolation of the nuclear envelope and nuclear lamin to the ring structures. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells' fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) serves as the sole bidirectional gateway of macromolecules in and out of the nucleus. Owing to its size and complexity (∼1,000 protein subunits, ∼110 MDa in humans), the NPC has remained one of the foremost challenges for structure determination. Structural studies have now provided atomic-resolution crystal structures of most nucleoporins. The acquisition of these structures, combined with biochemical reconstitution experiments, cross-linking mass spectrometry, and cryo–electron tomography, has facilitated the determination of the near-atomic overall architecture of the symmetric core of the human, fungal, and algal NPCs. Here, we discuss the insights gained from these new advances and outstanding issues regarding NPC structure and function. The powerful combination of bottom-up and top-down approaches toward determining the structure of the NPC offers a paradigm for uncovering the architectures of other complex biological machines to near-atomic resolution. [ABSTRACT FROM AUTHOR]

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes. [ABSTRACT FROM AUTHOR]

The article describes nuclear pore complexes (NPCs) as large protein assemblies in the nuclear envelope acting as portals for protein and RNA transport between the nucleus and cytoplasm. It notes that the NPC protein inventory, the nucleoporins, has been studied for diverse eukaryotes. It adds that the nucleoporins forming the Y-complex do not show an equal stoichiometry, unlike in other organisms.

Abstract Cell membranes of biological cells embedded with nuclear pore complexes (NPCs) are capable of controlling the flow of ions, e.g. Na+, K+ and Ca2+ by responding to stimuli, e.g. pH and voltage. From the inspiration of NPCs, researchers have been endeavoring to develop nanogates to achieve the control of ion transport, but the developed nanogates only have a low selectivity, the factor of change (FC) in ion fluxes due to ON/OFF switching. To our knowledge, nanogates with high responsiveness to temperature changes have not been reported. As such membranes etched with nanopores having a high selectivity (FC) and the reversible gating of temperature, pH and voltage were to be developed in the work. The nanogates were modified with DNA strands, whose linkers, the six nucleobases at 3′ ends, are complementary. According to the experimental results, at mildly acidic pH values, e.g. pH 5.3, the prepared nanogate is in an open state, while at neutral pH values, e.g. pH 7.9, it is shut off. The change in ion fluxes is up to a factor of 90, which is remarkably high compared to other nanogates reported. It is observed experimentally that promoting the number of the complementary nucleobases at the 3′ ends of the strands would improve nanogate performance significantly, while varying the other nucleobases exerts little effects. Hence the formation of nucleobase pairs among the complementary nucleobases at the 3′ ends of the strands leads to the binding of various strands, self-assembly of a strand web and blockage of ion transport. When the temperature is increased, due to the promotion of the thermal motion of DNA strands, the nucleobase pairs and the strand web will not be generated and the nanogate will remain open even at pH 7.9. Hence the nanogate developed is capable of responding to temperature changes. Further experiments were performed to investigate the influence of the NaCl concentrations and small opening diameters exerted on nanogate performance. Graphical abstract Image 1 Highlights • Nanogates reported only have a low selectivity, the factor of change in ion currents due to ON/OFF switching. • The nanogates of the membrane were modified with DNA strands, whose linkers are six nucleobases at 3′ ends. • The membrane developed is capable of responding to temperature changes, in addition to pH and voltage changes. • The change in ion fluxes is up to a factor of 90, remarkably high compared to other nanogates reported. • The formation of nucleobase pairs causes the self-assembly of a strand web and blockage of ion transport. [ABSTRACT FROM AUTHOR]

A microscopic transport model of a polymer translocating through a nuclear pore complex (NPC) is presented based on self-consistent field theory (SCFT), with the NPC and its nucleoporins mimicked by a hairy channel. Multiple cell environment effects (electrolyte effect, excluded volume effect, NPC drag effect, and hydrophobic effect) are all considered in this hairy channel model. The influence of various parameters (polymer chain length, length of NPC, strength of hydrophobic effect, and excluded volume effect) on translocation time is studied through theoretical analysis and numerical calculation. Numerical simulation results show that an area of low nucleoporin number density exists in the NPC, which facilitates the translocation of the polymer. The results also show that the translocation time curves with increasing NPC length and polymer charge number are concave. In addition, there are critical values for NPC length and polymer charge number for which the translocation time has a minimal value. The translocation time decreases with the increasing strength of the hydrophobic effect and excluded volume effect. [ABSTRACT FROM AUTHOR]

Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by "Live CLEM" imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in postmitotic NPC formation, we used artificial beads conjugated with anti‐GFP antibody, which captures GFP‐fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133‐coated beads or Nup153‐coated beads showed that Nup133 and Nup153, as the sole effector molecules, assembled the NPC‐like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133‐coated beads showed that Nup133 effectively assembled Nup107 and ELYS, whereas minimal assembly of Nup98 and Nup62 was observed; the Nup153‐coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange. They are exceptionally large protein complexes that fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. About 30 different protein components, termed nucleoporins, assemble in multiple copies into an intricate cylindrical architecture. Here, we review our current knowledge of the structure of nucleoporins and how those come together in situ. We delineate architectural principles on several hierarchical organization levels, including isoforms, posttranslational modifications, nucleoporins, and higher-order oligomerization of nucleoporin subcomplexes. We discuss how cells exploit this modularity to faithfully assemble NPCs. [ABSTRACT FROM AUTHOR]

Abstract The nuclear pore membrane protein 121 (POM121) was originally thought to be a constitutive protein of the nuclear pore complex (NPC). In addition to being involved in NPC assembly, abnormal POM121 expression has been found to be associated with many diseases. In this study, we explored, in detail, the effect of POM121 on the macrophage inflammatory response and found that its expression was significantly lower in LPS-stimulated macrophages, substantially amplifying pro-inflammatory cytokine (TNF-α and IL-6) production, suggesting that POM121 exerts a potent inhibitory effect on macrophage inflammation. Consistent with this notion, greater susceptibility to LPS-induced acute lung injury (ALI) as well as more severe tissue inflammation were found in POM121fl/fl Lyzm-Cre+ mice compared to those in control mice, as evidenced by the more severe lung injury and inflammation, increased TNF-α and IL-6 production and more abundant proteins in bronchoalveolar lavage fluid (BALF). This inflammation-modulating effect of POM121 relied on its ability to repress the NF-κB signal pathway via inhibition of phosphorylated P65 (phos-P65) nuclear accumulation. In the present study, we reported that in addition to acting as a constitutive NPC component, POM121 also modulated LPS-induced macrophage inflammation via repressing nuclear P65 translocation. Our study may pave the way for regulating LPS-induced massive macrophage inflammation and providing evidence for the functional diversity of nucleoporins (Nups). Highlights • POM121 is down-regulated in LPS-stimulated macrophages. • POM121 inhibits the macrophage inflammatory response. • Macrophage POM121 deficient mice show more susceptibility to LPS-inducted acute lung injury. • POM121 selectively inhibits nuclear accumulation of NF-κB p65. [ABSTRACT FROM AUTHOR]

Nucleoporins are a specialized subset of nuclear proteins that comprise the nuclear pore complex and regulate nucleocytoplasmic transport. Recent demonstrations of roles for individual nucleoporins in multiple paradigms of differentiation via mechanisms independent of nuclear trafficking represent conceptual advances in understanding the contributions of nucleoporins to cellular development. Among these, a functional role for nucleoporins in reproductive fitness and gametogenesis has been identified, supported by robust models and clinical studies that leverage the power of next generation sequencing technology to identify reproductive-disease-associated mutations in specific nucleoporins. Proper nucleoporin function manifests in different ways during oogenesis and spermatogenesis. However, nonhuman models of gametogenesis may not recapitulate human mechanisms, which may confound translational interpretation and relevance. To circumvent these limitations, identification of reproductive pathologies in patients, combined with next generation sequencing approaches and advanced in silico tools, offers a powerful approach to investigate the potential function of nucleoporins in human reproduction. Ultimately, elucidating the role of nucleoporins in reproductive biology will provide opportunities for predictive, diagnostic, and therapeutic strategies to address reproductive disorders. [ABSTRACT FROM AUTHOR]

Highlights • The nuclear envelope resists quantitative study by conventional light microscopy. • Super-resolution images contain sufficient data to allow precise numerical analysis. • An algorithm to quantify nuclear pore complex and lamina distribution is described. • The algorithm can be easily adapted to the analysis of other cellular components. Abstract For decades, components of the mammalian nuclear envelope (NE), such as the nuclear lamina and nuclear pore complexes (NPCs), have been largely resistant to quantitative cell biological analysis using conventional fluorescence microscopy. This is in part due to their sub diffraction limit dimensions. Super-resolution microscopy, a major advancement in cell biology research, has now made possible the acquisition of images in which nuclear lamin networks and single NPCs can be resolved in intact mammalian somatic cells. In particular, single molecule localization microscopy is able to generate data sets that are accurate enough to allow detailed quantitative analysis. Here we describe an algorithm that will identify the centroid of single NPCs and will determine their localization relative to the distribution of lamin protein filaments. Using this algorithm, a percentage of NPCs localized within the nuclear lamin network was accurately calculated, that could be compared between cells expressing different lamin complements. With modifications tweaked according to user specified sample images, this algorithm serves as a semi-automatic and fast computational tool to quantify and compare the localization and distribution of two or more cellular components at the nanometre scale. [ABSTRACT FROM AUTHOR]

Nuclear pore complex (NPC) is a fundamental component of the nuclear envelope and is key to the nucleocytoplasmic transport. Mutations in several NUP genes that encode individual components of NPC known as nucleoporins have been identified in recent years among patients with static encephalopathies characterized by developmental delay and microcephaly. We describe a multiplex consanguineous family in which four affected members presented with severe neonatal hypotonia, profound global developmental delay, progressive microcephaly and early death. Autozygome and linkage analysis revealed that this phenotype is linked to a founder disease haplotype (chr9:127,113,732-135,288,807) in which whole exome sequencing revealed the presence of a novel homozygous missense variant in NUP214. Functional analysis of patient-derived fibroblasts recapitulated the dysmorphic phenotype of nuclei that was previously described in NUP214 knockdown cells. In addition, the typical rim staining of NUP214 is largely displaced, further supporting the deleterious effect of the variant. Our data expand the list of NUP genes that are mutated in encephalopathy disorders in humans. [ABSTRACT FROM AUTHOR]

Nuclear pore complex (NPC) is the largest multimeric protein assembly of the eukaryotic cell, which mediates the nucleocytoplasmic transport. The constituent proteins of this assembly (nucleoporins) are present in varying copy numbers to give a size from ~ 60 MDa (yeast) to 112 MDa (human) and share common ancestry with other membrane‐associated complexes such as COPI/COPII and thus share the same structural folds. However, the nucleoporins across species exhibit very low percentage sequence similarity and this reflects in their distinct secondary structure and domain organization. We employed thorough sequence and phylogenetic analysis guided from structure‐based alignments of all the nucleoporins from fungi to metazoans to understand the evolution of NPC. Through evolutionary pressure analysis on various nucleoporins, we deduced that these proteins are under differential selection pressure and hence the homologous interacting partners do not complement each other in the in vitro pull‐down assay. The super tree analysis of all nucleoporins taken together illustrates divergent evolution of nucleoporins and notably, the degree of divergence is more apparent in higher order organisms as compared to lower species. Overall, our results support the hypothesis that the protein–protein interactions in such large multimeric assemblies are species specific in nature and hence their structure and function should also be studied in an organism‐specific manner. [ABSTRACT FROM AUTHOR]

The partitioning of the proteome between nucleus and cytoplasm affects nearly every aspect of eukaryotic biology. Despite this central role, we still have a poor understanding of which proteins localize in the nucleus and how this varies in different cell types and conditions. Recent advances in quantitative proteomics and high-throughput imaging are starting to close this knowledge gap. Studies on protein interaction are beginning to reveal the spectrum of cargos of nuclear import and export receptors. We anticipate that it will soon be possible to predict each protein's nucleocytoplasmic localization based on its importin/exportin interactions and its estimated diffusion rate through the nuclear pore. This insight is likely to provide us with a fundamental understanding of how cells use nucleocytoplasmic partitioning to encode and relay information. [ABSTRACT FROM AUTHOR]

Graphical abstract Nuclear export of individual mRNAs through nuclear pore complexes revealed by SPEED microscopy. Abstract The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs. In this review, we firstly evaluate the necessity of single-molecule live-cell microscopy in the study of mRNA nuclear export. Then, we highlight the application of single-point edge-excitation sub-diffraction (SPEED) microscopy that combines high-speed SMF microscopy and a 2D-to-3D transformation algorithm in the studies of nuclear transport kinetics and route for mRNAs. Finally, we summarize the new features of mRNA nuclear export found with SPEED microscopy as well as the reliability and accuracy of SPEED microscopy in mapping the 3D spatial locations of transport routes adopted by proteins and mRNAs through the NPCs. [ABSTRACT FROM AUTHOR]

Key message: Loss-of-function of nucleoporin NUP1 in Arabidopsis causes defect in both male and female gametogenesis. Its ovules are arrested during meiosis, and its pollen grains are aborted at mitosis I.Abstract: Nuclear pore complex (NPC) plays crucial roles in nucleocytoplasmic trafficking of proteins and RNAs. The NPC contains approximately 30 different proteins termed nucleoporins (NUPs). So far, only a few of plant NUPs have been characterized. The Arabidopsis NUP1 was identified as an ortholog of the yeast NUP1 and animal NUP153. Loss-of-function of NUP1 in Arabidopsis caused fertility defect; however, the molecular mechanism of this defect remains unknown. Here, we found that both male and female gametogenesis of the nup1 mutants were defective. nup1 ovules were arrested from the meiosis stage onward; only approximately 6.7% and 3% ovules of the nup1-1 and nup1-4 mutants developed up to the FG7 stage, respectively. Pollen development of the nup1 mutants was arrested during the first mitotic division. In addition, enlarged pollen grains with increased DNA content were observed in the nup1 mutant. RNA-sequencing showed that expression levels of genes involved in pollen development or regulation of cell size were reduced dramatically in nup1 compared with wild type. These results suggest that NUP1 plays an important role in gametogenesis. [ABSTRACT FROM AUTHOR]

In biological cells, nuclear pore complexes (NPCs) embedded in cell membranes are capable of controlling the flow of ions, for example, Na+, K+, and Ca2+ by responding to stimuli, for example, pH and voltage. Inspired by NPCs, researchers have been endeavoring to develop nanogates to achieve the control of ion transport, but the developed nanogates only have a low factor of change in ion currents due to ON/OFF switching. As such nanopores with high temperature and pH responsivities were developed in this work. According to the experimental results, at a voltage of 3 V, the change in ion currents due to pH change is up to a factor of 170, which is remarkably high compared to other nanogates reported. Quantum chemical (QC) calculation results show that a protonated cytosine molecule (C+) and an unprotonated cytosine molecule (C) form three pairs of hydrogen bonds and consequently a nucleobase pair, CC+, leading to the binding of various strands, assembly of a strand net, and blockage of ion transport. The nanogate developed is capable of responding to temperature change. At a voltage of 3 V, the factor of change in ion currents in response to temperature variation is as high as 110. Further experiments were performed to investigate the influence of the NaCl concentrations and small opening diameters exerted on nanogate performance. Inspired by nuclear pore complexes (NPCs), researchers have been endeavoring to develop nanogates to achieve the control of ion transport, but developed nanogates only have a low factor of change in ion currents due to ON/OFF switching. Nanopores with high temperature and pH responsivities were developed in the work. [ABSTRACT FROM AUTHOR]

The nuclear pore complex is the main transportation hub for exchange between the cytoplasm and the nucleus. It is built from nucleoporins that form distinct subcomplexes to establish this huge protein complex in the nuclear envelope. Malfunctioning of nucleoporins is well known in human malignancies, such as gene fusions of NUP214 and NUP98 in hematological neoplasms and overexpression of NUP88 in a variety of human cancers. In the past decade, the incremental utilization of next-generation sequencing has unraveled mutations in nucleoporin genes in the context of an increasing number of hereditary diseases, often in a tissue-specific manner. It emerges that, on one hand, the central nervous system and the heart are particularly sensitive to mutations in nucleoporin genes. On the other hand, nucleoporins forming the scaffold structure of the nuclear pore complex are eminently mutation-prone. These novel and exciting associations between nucleoporins and human diseases emphasize the need to shed light on these unanticipated tissue-specific roles of nucleoporins that may go well beyond their role in nucleocytoplasmic transport. In this review, the current insights into altered nucleoporin function associated with human hereditary disorders will be discussed. [ABSTRACT FROM AUTHOR]

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4+ T cells demonstrated that multiple components of the nuclear pore complex and nuclear import machinery can impact MX2 anti-viral activity. In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1. [ABSTRACT FROM AUTHOR]

Abstract Nuclear pore complex (NPC) is a biomolecular "nanomachine" that controls nucleocytoplasmic transport in eukaryotic cells. The key component of the functional architecture of the NPC is the assembly of intrinsically disordered proteins that line its passageway and play a central role in the NPC transport mechanism. Due to paucity of experimental methods capable to directly probe the morphology of this assembly in intact NPCs, much of our knowledge about its properties derives from in vitro experiments augmented by theoretical and computational modeling. I review the major insights into the biophysics of the assemblies of the intrinsically disordered proteins of the NPC arising from the theoretical analysis of the recent in vitro experimental results, with the emphasis on the phase separation and aggregation phenomena. Graphical Abstract Unlabelled Image Highlights • Nuclear pore complex (NPC) is a biomolecular "nanomachine" that controls nucleocytoplasmic transport in eukaryotic cells. • The key component of the functional architecture of the NPC is the assembly of intrinsically disordered proteins that line its passageway. • Much of our knowledge about its properties derives from in vitro experiments on the assemblies of these intrinsically disordered proteins. • Theoretical and computational analysis of the experimental findings based on the statistical physics has pinpointed the key factors responsible for the spatial organization of the assemblies of the intrinsically disordered proteins of the NPC. [ABSTRACT FROM AUTHOR]

Lamins are the main component of the nuclear lamina, a protein meshwork at the inner nuclear membrane which primarily provide mechanical stability to the nucleus. Lamins, type V intermediate filament proteins, are also involved in many nuclear activities. Structural analysis of nuclei revealed that lamins form 3.5 nm thick filaments often interact with nuclear pore complexes. Mutations in the LMNA gene, encoding A-type lamins, have been associated with at least 15 distinct diseases collectively termed laminopathies, including muscle, metabolic and neurological disorders, and premature aging syndrome. It is unclear how laminopathic mutations lead to such a wide array of diseases, essentially affecting almost all tissues. [ABSTRACT FROM AUTHOR]

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype. [ABSTRACT FROM AUTHOR]

Highlights • Recent cryo-EM structures reveal similarities and differences between membrane-binding coats of nuclear pore complex and coated vesicles. • The nuclear pore complex scaffold can be conceptualized as an insertion of a COPII coat into a COPI coat. • In situ cryo-ET will be an important tool for understanding evolution. Nuclear pores and coated vesicles are elaborate multi-component protein complexes that oligomerize on membranes, and stabilize or induce membrane curvature. Their components, nucleoporins and coat proteins, respectively, share similar structural folds and some principles of how they interact with membranes. The protocoatomer hypothesis postulates that this is due to divergent evolution from a common ancestor. It therefore has been suggested that nucleoporins and coat proteins have similar higher order architectures. Here, we review recent work that relied on technical advances in cryo-electron microscopy and integrative structural biology to take a fresh look on how these proteins form membrane coats in situ. We discuss the relationship between the architectures of nuclear pores and coated vesicles, and their evolutionary origins. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) displays the unique ability to infect nondividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect nondividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not nondividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin. [ABSTRACT FROM AUTHOR]

Metformin has been a frontline therapy for type 2 diabetes (T2D) for many years. Its effectiveness in T2D treatment is mostly attributed to its suppression of hepatic gluconeogenesis; however, the mechanistic aspects of metformin action remain elusive. In addition to its glucose-lowering effect, metformin possesses other pleiotropic health-promoting effects that include reduced cancer risk and tumorigenesis. Metformin inhibits the electron transport chain (ETC) and ATP synthesis; however, recent data reveal that metformin regulates AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin complex 1 (mTORC1) by multiple, mutually nonexclusive mechanisms that do not necessarily depend on the inhibition of ETC and the cellular ATP level. In this review, we discuss recent advances in elucidating the molecular mechanisms that are relevant for metformin use in cancer treatment. Highlights Metformin use is associated with lower cancer incidence in diabetic patients and is considered for cancer therapy of non-diabetic patients. Metformin inhibits ETC and elevates AMP/ATP ratio, resulting in AMPK activation and mTORC1 inhibition. In addition, at metformin concentrations that do not affect the ETC and AMP/ATP ratio, administration of metformin results in AMPK activation and mTORC1 inhibition by mechanisms not involving ETC. Metformin-induced inhibition of ATP synthesis affects the nuclear pore complex, which leads to nuclear exclusion of RagC. Without being able to pass through the nucleus, RagC cannot be potentiated for mTORC1 activation. [ABSTRACT FROM AUTHOR]

Export of mRNAs from the nucleus to the cytoplasm is a key regulatory step in the expression of proteins. mRNAs are transported through the nuclear pore complex (NPC). Export of mRNAs responds to a variety of cellular stimuli and stresses. Revelations of the specific effects elicited by NPC components and associated co-factors provides a molecular basis for the export of selected RNAs, independent of bulk mRNA export. Aberrant RNA export has been observed in primary human cancer specimens. These cargo RNAs encode factors involved in nearly all facets of malignancy. Indeed, the NPC components involved in RNA export as well as the RNA export machinery can be found to be dysregulated, mutated, or impacted by chromosomal translocations in cancer. The basic mechanisms associated with RNA export with relation to export machinery and relevant NPC components are described. Therapeutic strategies targeting this machinery in clinical trials is also discussed. These findings firmly position RNA export as a targetable feature of cancer along with transcription and translation.

Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution. [Display omitted] • A composite multiscale 3D structure of the isolated yeast NPC has been determined • Double nuclear outer rings represent an ancient feature of NPCs • Membrane anchor sites on the inner ring use a nucleoporin β-propeller/TMD partner motif • Radial expansion of the NPC scaffold unplugs the central channel to promote transport Akey et al. report a multiscale structure of the isolated yeast NPC that provides a framework to piece together the roles of nucleoporins in assembly and transport while providing insights into the evolution of critical sub-assemblies. [ABSTRACT FROM AUTHOR]