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Separation of DNA replication and transcription, which occur in the nucleus, from protein synthesis, which occurs in the cytoplasm, allows a more precise regulation of these processes. Selective exchange of macromolecules between the two compartments is mediated by proteins of the nuclear pore complex (NPC). Receptor proteins of the karyopherin family interact with NPC components and transfer their cargos between the nucleus and cytoplasm. Nucleocytoplasmic transport pathways are regulated at multiple levels by modulating the expression or function of individual cargoes, transport receptors, or the transport channel. The regulatory levels have increasingly broad effects on the transport pathways and affect a wide range of processes from gene expression to development and differentiation. [ABSTRACT FROM AUTHOR]

Cell death requires coordinated intracellular signalling before disassembly of cell architecture by degradative enzymes. Although the death signalling cascades that involve the mitochondria, the ER and the plasma membrane have been extensively characterized, only a handful of studies have examined the functional and structural alterations of the nuclear pore complex (NPC) during neuronal death. Here, we show that during excitotoxic neuronal degeneration calpains redistributed across the nuclear envelope and mediated the degradation of NPC components causing altered permeability of the nuclear membrane. In primary dissociated neurons, simultaneous recording of cytosolic [Ca2+] and localization of fluorescent proteins showed that the onset of Ca2+ overload signalled a progressive increase in the diffusion of small reporter molecules across the nuclear envelope. Later, calpain-mediated changes in nuclear pore permeability allowed accumulation of large proteins in the nucleus. Further, in a model of excitotoxic neuronal degeneration in Caenorhabditis elegans, we found similar nuclear changes and redistribution of fluorescent probes across the nuclear membrane in dying neurons. Our findings strongly suggest that increased leakiness of the nuclear barrier affects nucleocytoplasmic transport, alters the localization of proteins across the nuclear envelope and it is likely to be involved in Ca2+-dependent cell death, including ischemic neuronal demise. [ABSTRACT FROM AUTHOR]

Suppression of a nuclear pore protein Nup358/RanBP2 is linked to mitotic cell death, but the clinical relevance of this link is unknown. In a recent issue of Cell , Vecchione et al. (2016) show that in approximately 10% of BRAF-like colorectal cancer (CC) patients, Nup358/RanBP2 is critical for survival. Treatment with vinorelbine, a microtubule-depolymerizing drug that inhibits mitosis, might be a potential treatment for these CCs. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are 110-megadalton assemblies that mediate nucleocytoplasmic transport. NPCs are built from multiple copies of ~30 different nucleoporins, and understanding how these nucleoporins assemble into the NPC scaffold imposes a formidable challenge. Recently, it has been shown how the Ycomplex, a prominent NPC module, forms the outer rings of the nuclear pore. However, the organization of the inner ring has remained unknown until now. We used molecular modeling combined with cross-linking mass spectrometry and cryo-electron tomography to obtain a composite structure of the inner ring. This architectural map explains the vast majority of the electron density of the scaffold. We conclude that despite obvious differences in morphology and composition, the higher-order structure of the inner and outer rings is unexpectedly similar. [ABSTRACT FROM AUTHOR]

The nuclear pore complexes are complex protein structures located in the nuclear envelope, where they control the nuclear-cytoplasmic transport, and inside the stacks of endoplasmic reticulum cisternae, annulate lamellae. After overexpression of some nucleoporins, numerous granules are visible in the cytoplasm. According to the published data, these granules are the annulate lamellae. In the current paper, the structural organization of POM121-containing granules was analyzed using correlative light and electron microscopy. The ultrastructural study demonstrates that POM121-containing granules are not annulate lamellae but aggregates of endoplasmic reticulum membranes. Thus, overexpressed POM121 is not able to induce the annulate lamella formation. The mechanisms of self-organization of non-functional structures (such as the aggregates of endoplasmic reticulum membranes described here) and possible involvement of these mechanisms in the formation of cellular structures are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The developmental processes that give rise to the animal body plan are exceedingly complex. Model systems such as Drosophila melanogaster have yielded profound insight into roles of conserved genes and genetic pathways in development. Drosophila development begins with the formation of sperm and eggs, and proceeds through several morphologically distinct stages including development of the early embryo, larval instars, formation of pupae, and differentiation of adult tissues. The nuclear transport of proteins and RNAs represents a critical step in the regulation of gene expression during every stage of development and tissue differentiation. Studies of the nuclear transport machinery in Drosophila refute the notion that nuclear transport is strictly a housekeeping process without specific regulatory roles in development. Rather, they support the idea that the basal nuclear transport machinery has adapted during the evolution of the metazoan body plan to play important regulatory roles in key developmental events. [Copyright &y& Elsevier]

Abstract: Nucleocytoplasmic trafficking of macromolecules, a highly specific and tightly regulated process, occurs exclusively through the nuclear pore complex. This immense structure is assembled from approximately 30 proteins, termed nucleoporins. Here we discuss the four nucleoporins that have been linked to cancers, either through elevated expression in tumors (Nup88) or through involvement in chromosomal translocations that encode chimeric fusion proteins (Tpr, Nup98, Nup214). In each case we consider the normal function of the nucleoporin and its translocation partners, as well as what is known about their mechanistic contributions to carcinogenesis, particularly in leukemias. Studies of nucleoporin-linked cancers have revealed novel mechanisms of oncogenesis and in the future, should continue to expand our understanding of cancer biology. [Copyright &y& Elsevier]

Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis. [ABSTRACT FROM AUTHOR]

Although the separation of transcription and translation, mediated by the nuclear envelope, is the defining characteristic of Eukaryotes, the barrier between the nuclear and cytoplasmic compartments needs to be semipermeable to enable material to be moved between them. Moreover, each compartment needs to have a distinctive complement of macromolecules to mediate specific functions and so movement between them needs to be controlled. This is achieved through the selective active transport of macromolecules through the nuclear pores that stud the nuclear envelope, and which serve as a conduit between these compartments. Nuclear pores are huge cylindrical macromolecular assemblies and are constructed from the order of 30 different proteins called nucleoporins. Nuclear pores have a central transport channel that is filled with a dense network of natively unfolded portions of many different nuclear pore proteins (nucleoporins or nups). This network generates a barrier that impedes, but does not entirely prevent, the diffusion of many macromolecules through the pores. The rapid movement of a range of proteins and RNAs through the pores is mediated by a range of transport factors that bind their cargo in one compartment and release it in the other. However, although as their size increases the diffusion of macromolecules through nuclear pores is progressively impaired, additional mechanisms, including the binding of some macromolecules to immobile components of each compartment and also the active removal of macromolecules from the inappropriate compartment, are needed to fully maintain the distinctive compositions of each compartment. [ABSTRACT FROM AUTHOR]

Abstract: A novel organelle-like membrane specialization has been found in an epithelial cell line. Characteristically, the helical membrane arrays (referred to hereafter as TUHMAs) are organized around tubular, proteinaceous electron-dense cores of 80 nm in diameter. Depending on the cell status, up to 8 of these cores provide the basis for an intermingled membrane scaffold of an overall length of 3–5 μm. TUHMAs exist as single organelles in transient association with the nucleus, the rough endoplasmic reticulum, patches of annulate lamellae, and the Golgi complex. While most of the constituents are still unknown, evidence for an involvement of nucleoporins in TUHMA organization is presented, as shown by fluorescence immunochemistry. This should make TUHMAs more easily accessible for future studies on their structure and function. [Copyright &y& Elsevier]

The gene regulation mechanisms underlying hormone- and light-induced signal transduction in plants rely not only on post-translational modification and protein degradation, but also on selective inclusion and exclusion of proteins from the nucleus. For example, plant cells treated with light or hormones actively transport many signalling regulatory proteins, transcription factors, and even photoreceptors and hormone receptors into the nucleus, while actively excluding other proteins. The nuclear envelope (NE) is the physical and functional barrier that mediates this selective partitioning, and nuclear transport regulators transduce hormone- or light-initiated signalling pathways across the membrane to mediate nuclear activities. Recent reports revealed that mutating the proteins regulating nuclear transport through the pores, such as nucleoporins, alters the plant's response to a stimulus. In this review, recent works are introduced that have revealed the importance of regulated nucleocytoplasmic partitioning. These important findings deepen our understanding about how co-ordinated plant hormone and light signal transduction pathways facilitate communication between the cytoplasm and the nucleus. The roles of nucleoporin components within the nuclear pore complex (NPC) are also emphasized, as well as nuclear transport cargo, such as Ran/TC4 and its binding proteins (RanBPs), in this process. Recent findings concerning these proteins may provide a possible direction by which to characterize the regulatory potential of hormone- or light-triggered nuclear transport. [ABSTRACT FROM PUBLISHER]

Abstract: Phenylalanine–glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-β. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-β, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-β-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG–receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG–receptor interactions instead of FG–FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force–volume (FV) mode. [Copyright &y& Elsevier]

Abstract: The nuclear pore complex (NPC) is the only known gateway for exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. One key compound of the NPC is the p62 subcomplex, which consists of the nucleoporins p62, p54, and p58/p45 and is supposed to be involved in nuclear protein import and export. Here we show the localization of distinct domains of the p62 complex by immuno-electron microscopy using isolated nuclei from Xenopus oocytes. To determine the exact position of the p62 complex, we examined the localization of the C and N-terminal domains of p62 by immunogold-labeling using domain-specific antibodies against p62. In addition we expressed epitope-tagged versions of p62, p54, and p58 in Xenopus oocytes and localized the domains with antibodies against the tags. This first systematic analysis of the domain topology of the p62 complex within the NPC revealed that the p62 complex is anchored to the cytoplasmic face of the NPC most likely by the coiled-coil domains of the three nucleoporins. Furthermore, we found the phenylalanine-glycine (FG)-repeat domain of p62, but not of p58 and p54, to be of mobile and flexible nature. [Copyright &y& Elsevier]

FXR proteins are part of ribonucleoprotein granules controlling stability, translation, and cellular localization of target mRNAs. In the current issue, Agote‐Aran et al extend the repertoire of FXR protein action and show that they are also involved in the transport of nuclear pore complex components to the nuclear envelope and thereby prevent cytoplasmic aggregation of these proteins. [ABSTRACT FROM AUTHOR]

Selective autophagy is critical for the regulation of cellular homeostasis in organisms from yeast to humans. This process is a specific degradation pathway for a wide variety of substrates including unwanted cytosolic components, such as protein aggregates, damaged and/or superfluous organelles, and pathogens. However, it has been less clear as to whether a protein complex or substructure of an organelle can be targeted for removal by selective autophagy. One example of such a substrate is the nuclear pore complex (NPC), a large macromolecular assembly that is present throughout the nuclear envelope. Here, we highlight two recent studies that demonstrate for the first time that NPCs are targeted for vacuolar degradation through selective autophagy. AIM: Atg8-interacting motif; NE: nuclear envelope; NPC: nuclear pore complex; Nup: nucleoporin; PMN/micronucleophagy: piecemeal microautophagy of the nucleus [ABSTRACT FROM AUTHOR]

Abstract: In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-β. Crystallization studies by others have indicated that nuclear carriers such as importin-β bind to phenylalanine–glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-β identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-β. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-β binding to those repeats. In addition to these expected binding sequences for importin-β, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-β, indicating that additional Nup sequences may form binding sites for importin-β. [Copyright &y& Elsevier]

Nuclear pore complexes (NPCs) are the gateways for both active and passive bidirectional molecular transport between the nucleoplasm and cytoplasm. These mega-dalton assemblies are composed of multiple copies of approximately 30 distinct proteins termed nucleoporins. Higher eukaryotes display an “open” mitosis in which the NPCs, nuclear envelope, and lamina disassemble. During mitosis several nucleoporins are redistributed to kinetochores until they are recruited back to the periphery of chromatin as the NPCs are reassembled. Within this study we have developed and optimized the visualization of mammalian cells and their chromosome profiles throughout the cell-cycle. Close attention has been paid to the preservation of chromatin, membranes, and NPC structure to investigate the ultrastructural locations of specific proteins in both interphase and mitosis. [ABSTRACT FROM AUTHOR]

Abstract: Nucleoporins are components of the nuclear pore, which is required for nucleo-cytoplasmic transport. We report a role for a subclass of nucleoporins in orienting the mitotic spindle in C. elegans embryos. RNAi-mediated depletion of any of five putative nucleoporins npp-1, npp-3, npp-4, npp-11, and npp-13 leads to indistinguishable spindle orientation defects. Transgenic worms expressing NPP-1::GFP or NPP-11::GFP show GFP localization at the nuclear envelope, consistent with their predicted function. NPP-1 interacts with the other nucleoporins in yeast two-hybrid assays, suggesting that the proteins affect spindle orientation by a common process. The failed orientation phenotype of npp-1(RNAi) is at least partially epistatic to the ectopic spindle rotation in the AB blastomere of par-3 mutant embryos. This suggests that NPP-1 contributes to the mechanics of spindle orientation. However, NPP-1 is also required for PAR-6 asymmetry at the two-cell stage, indicating that nucleoporins may be required to define cortical domains in the germ line blastomere P1. Nuclear envelope structure is abnormal in npp-1(RNAi) embryos, but the envelope maintains its integrity, and most nuclear proteins we assayed accumulate normally. These findings raise the possibility that these nucleoporins may have direct roles in orienting the mitotic spindle and the maintenance of cell polarity. [Copyright &y& Elsevier]

Abstract: The death of yeast treated with hydrogen peroxide (H2O2) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H2O2-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H2O2-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H2O2-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead. [Copyright &y& Elsevier]

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH2- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1 . Thus, we propose that two independent translocation pathways cooperate to determine the steady- state distribution of Stat1. [ABSTRACT FROM AUTHOR]

Interactions with nucleoporins containing FxFG‐repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here the 1.9 Å resolution crystal structure of yeast NTF2‐N77Y bound to a FxFG‐nucleoporin core, which provides a basis for understanding this interaction and its role in nuclear trafficking. The two identical FxFG binding sites on the dimeric molecule are formed by residues from each chain of NTF2. Engineered mutants at the interaction interface reduce the binding of NTF2 to nuclear pores and cause reduced growth rates and Ran mislocalization when substituted for the wild‐type protein in yeast. Comparison with the crystal structure of FG‐nucleoporin cores bound to importin‐β and TAP/p15 identified a number of common features of their binding sites. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking. [ABSTRACT FROM AUTHOR]

Nucleo-cytoplasmic transport of proteins is mediated by nuclear export signals, identified in various proteins executing heterologous biological functions. However, the molecular mechanism underlying the orchestration of export is only poorly understood. Using microinjection of defined recombinant export substrates, we now demonstrate that leucine-rich nuclear export signals varied dramatically in determining the kinetics of export in vivo. Thus, nuclear export signals could be kinetically classified which correlated with their affinities for CRM1-containing export complexes in vitro. Strikingly, cotransfection experiments revealed that proteins containing a fast nuclear export signal inhibited export and the biological activity of proteins harboring a slower nuclear export signal in vivo. The affinity for export complexes seems therefore predominantly controlled by the nuclear export signal itself, even in the context of the complete protein in vivo. Overexpression of FG-rich repeats of nucleoporins affected a medium nuclear export signal containing protein to the same extent as a fast nuclear export signal containing protein, indicating that nucleoporins appear not to contribute significantly to nuclear export signal-specific export regulation. Our results imply a novel mode for controlling the biological activity of shuttle proteins already by the composition of the nuclear export signal itself. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A comprehensive model is presented of the yeast nuclear pore complex (NPC) • Connectors link together different structural and functional layers in the NPC • Multiple structural and functional NPC isoforms co-exist in each cell • Modular construction allows structural plasticity and inner ring dilation of the NPC A comprehensive model of the yeast NPC reveals an interconnected architecture of the core scaffold and provides an understanding of the isoforms and structural plasticity that may be associated with different functional states. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) mediate the selective and highly efficient transport between the cytoplasm and the nucleus. They are embedded in the two membrane structure of the nuclear envelope at sites where these two membranes are fused to pores. A few transmembrane proteins are an integral part of NPCs and thought to anchor these complexes in the nuclear envelope. In addition, a number of nucleoporins without membrane spanning domains interact with the pore membrane. Here we review our current knowledge of how these proteins interact with the membrane and how this interaction can contribute to NPC assembly, stability and function as well as shaping of the pore membrane. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Amyloids are fibrillar protein aggregates with a cross-β structure and unusual features, including high resistance to detergent or protease treatment. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Several examples of nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human NUP153) are supposed to form amyloid fibrils. In this study, we demonstrated an ability of the human NUP58 nucleoporin to form amyloid aggregates in vivo and in vitro. Moreover, we found two forms of NUP58 aggregates: oligomers and polymers stabilized by disulfide bonds. Bioinformatic analysis revealed that all known orthologs of this protein are potential amyloids which possess several regions with conserved ability to aggregation. The biological role of nucleoporin amyloid formation is debatable. We suggest that it is a rather abnormal process, which is characteristic for many proteins implicated in phase separation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have used antibodies against different proteins of the nuclear pore complex to identify cytoplasmic annulate lamellae by immunocytochemistry at the light- and electron-microscopic level in various lines of cultured mammalian cells. Although annulate lamellae (AL) are seldom observed in some cell lines, they occur in large numbers in other lineages, also with cell-type-specific size distributions. AL are especially abundant and prominent in African green monkey kidney epithelial cells of line RC37 and bovine mammary gland epithelial cells of line BMGE. We have studied the distribution of AL in relation to other organelles by using double-label immunofluorescence microscopy and have demonstrated a significant relationship between AL and rough endoplasmic reticulum. We have further shown that AL are disassembled during prophase and reassembled at the end of mitosis, almost concomitantly with the disassembly and reassembly of the nuclear envelope. Cultured mammalian cells that are rich in AL can therefore be used as suitable models for studies of the biogenesis of these lamellae, which can conveniently be detected by immunofluorescence microscopy. [ABSTRACT FROM AUTHOR]

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.

In budding yeast, the Rho-family GTPase Cdc42 has several functions that depend on its subcellular localization and the cell cycle stage. During bud formation, Cdc42 localizes to the plasma membrane at the bud tip and bud neck where it carries out functions in actin polymerization, spindle positioning, and exocytosis to ensure proper polarity development. Recent live-cell imaging analysis revealed a novel localization of Cdc42 to a discrete intracellular focus associated with the vacuole and nuclear envelope. The discovery of this novel Cdc42 localization led to the identification of a new function in ESCRT-mediated nuclear envelope sealing. However, other aspects of this intracellular localization and its functional implications were not explored. Here, we further characterize the Cdc42 focus and present several novel observations that suggest possible additional Cdc42 functions at the nucleus, including nucleus-vacuole junction formation, nuclear envelope tethering, nuclear migration, and nucleopodia formation.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) where they ensure the transport of macromolecules between the nucleus and the cytoplasm. NPCs are built from nucleoporins (Nups) through a sequential assembly order taking place at two different stages during the cell cycle of mammalian cells: at the end of mitosis and during interphase. In addition, fragile X-related proteins (FXRPs) can interact with several cytoplasmic Nups and facilitate their localization to the NE during interphase likely through a microtubule-dependent mechanism. In the absence of FXRPs or microtubule-based transport, Nups aberrantly localize to the cytoplasm forming the so-called cytoplasmic nucleoporin granules (CNGs), compromising NPCs' function on protein export. However, it remains unknown if Nup synthesis or degradation mechanisms are linked to the FXRP-Nup pathway and if and how the action of FXRPs on Nups is coordinated with the cell cycle progression. Here, we show that Nup localization defects observed in the absence of FXR1 are independent of active protein translation. CNGs are cleared in an autophagy- and proteasome-independent manner, and their presence is restricted to the early G1 phase of the cell cycle. Our results thus suggest that a pool of cytoplasmic Nups exists that contributes to the NPC assembly specifically during early G1 to ensure NPC homeostasis at a short transition from mitosis to the onset of interphase., 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 © 2021 Agote-Arán, Lin and Sumara.)

The bi-directional nucleocytoplasmic shuttling of macromolecules like molecular signals, transcription factors, regulatory proteins, and RNAs occurs exclusively through Nuclear Pore Complex (NPC) residing in the nuclear membrane. This magnanimous complex is essentially a congregation of ~32 conserved proteins termed Nucleoporins (Nups) present in multiple copies and mostly arranged as subcomplexes to constitute a functional NPC. Nups participate in ancillary functions such as chromatin organization, transcription regulation, DNA damage repair, genome stabilization, and cell cycle control, apart from their central role as nucleocytoplasmic conduits. Thus, Nups exert a role in the maintenance of cellular homeostasis. In mammals, precisely three nucleoporins traverse the nuclear membrane, are called transmembrane Nups (TM-Nups), and are involved in multiple cellular functions. Owing to their vital roles in cellular processes and homeostasis, dysregulation of nucleoporin function is implicated in various diseases. The deregulated functioning of TM-Nups can thus act as an opportune window for the development of diseases. Indeed, mounting evidence exhibits a strong association of TM-Nups in cancer and numerous other physiological disorders. These findings have provided much-needed insights into the novel mechanisms of disease progression. While nucleoporin's functions have often been summarized in the disease context, a focus on TM-Nups has always lacked. This review emphasizes the elucidation of distinct canonical and non-canonical functions of mammalian TM-Nups and the underlying mechanisms of their disease association., 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 © 2021 Bindra and Mishra.)

Picornaviruses are positive-stranded RNA viruses. Even though replication and translation of their genome take place in the cytoplasm, these viruses evolved different strategies to disturb nucleocytoplasmic trafficking of host proteins and RNA. The major targets of picornavirus are the phenylalanine-glycine (FG)-nucleoporins, which form a mesh in the central channel of the nuclear pore complex through which protein cargos and karyopherins are actively transported in both directions. Interestingly, while enteroviruses use the proteolytic activity of their 2A protein to degrade FG-nucleoporins, cardioviruses act by triggering phosphorylation of these proteins by cellular kinases. By targeting the nuclear pore complex, picornaviruses recruit nuclear proteins to the cytoplasm, where they increase viral genome translation and replication; they affect nuclear translocation of cytoplasmic proteins such as transcription factors that induce innate immune responses and retain host mRNA in the nucleus thereby preventing cell emergency responses and likely making the ribosomal machinery available for translation of viral RNAs. [ABSTRACT FROM AUTHOR]

Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles. [Display omitted] • Incomplete mitotic NPC dissociation yields octameric inner and outer ring subassemblies • Octameric inner and outer ring subassemblies localize to fenestrated mitotic ER • Octameric inner and outer ring subassemblies template post-mitotic NPC formation • NPC's inherit octameric inner and outer ring subassemblies Chou et al. show that cells inherit inner and outer ring nuclear pore subassemblies from their great-great-great-great-great-great-great-great grandmothers. These "immortal" structures, retained during mitosis in highly fenestrated ER sheets, template post-mitotic nuclear pore complex (NPC) assembly as ER sheets recoat the chromatin masses in each of the two daughter cells. [ABSTRACT FROM AUTHOR]

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus. [Display omitted] • Co-translational interactions contribute to the assembly of NPCs • A subset of nuclear pore and nuclear proteins are translated at NPCs • Recognition of nascent Nup1/Nup2 by karyopherins is required for translation at NPCs • Localized translation of Nup1 prevents its aggregation in the cytoplasm Lautier et al. have systematically investigated the co-translational events targeting the assembly of the nuclear pore complex (NPC). They reveal that several nucleoporins establish binary interactions during translation in the cytoplasm and that a subset of them are translated at NPCs, their nascent chains being recognized by karyopherins. [ABSTRACT FROM AUTHOR]

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment. [ABSTRACT FROM AUTHOR]