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Visualization of NPC substructures. Scanning electron microscopy (left) of a vertebrate (Xenopus) NPC viewed en face from the cytoplasm best reveals the cytoplasmic filaments (CF); an NPC viewed similarly from the nucleoplasm shows the nuclear basket (NB). The structures of the central core are revealed by three-dimensional protein density maps generated by cryoelectron microscopy and image processing (CryoEM, right) of both vertebrate and yeast NPCs. The positions of the spoke (SP) and central transporter (T) are indicated on both the en face projection map (top row) and longitudinal slice (bottom row) of the vertebrate NPC. The positions of the cytoplasmic ring (CR), nuclear ring (NR), outer spoke-ring (OR), and inner spoke-ring (IR) are indicated on a longitudinal slice. Diagrams at the left of the micrographs show the corresponding orientation of the NPC. Micrographs were kindly provided by Martin Goldberg and Terry Allen (SEM) and Chris Akey (CryoEM). Bar, 50 nm.
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... Antimicrobial Agents and Chemotherapy SB7, and KF are not only the most complex isolates but also distinct, with polymorphisms mostly present in unique MS loci compared to the other isolates. The multiclonal isolates only share polymorphisms at three MS loci, AS12, AS15, and AS31, with only A12 present in the coding region of a nucleoporin protein [PF3D7_0609000 (60,61)], with A15 and AS31 in the untranslated region of proteins with unknown function (PF3D7_1364400 and PF3D7_0619100). The variation in genetic complexity between isolates was supported by the observed level of within-host genetic diversity of each isolate as described by the Fws index (the within-host infection fixation index), which revealed that the probability of recombina tion of different clones was lowest in SB2 and SB6 (Fws at 1 indicative of a clonal infection) due to the low complexity of infection. ...
Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum . Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.
... The central channel of the NPC is lined with intrinsically disordered nucleoporin proteins containing many phenylalanine/glycine (FG) motifs, i.e., FG-Nucleoporins (FG-Nups). These FG motifs have been shown to play an essential role in NTR-driven transport (1,2) and are also suggested to be important in maintaining the passive permeability barrier of the NPC (3)(4)(5). Various descriptions exist that relate the phase state of the FG-Nups in the NPC central channel to its selectivity: The selective phase model (6,7) proposes that the FG-Nups form a dense, sieve-like meshwork through specific FG-FG interactions that allows small particles to pass, while blocking larger molecules. ...
The intrinsically disordered FG-Nups in the central channel of the nuclear pore complex (NPC) form a selective permeability barrier, allowing small molecules to traverse by passive diffusion, while large molecules can only translocate with the help of nuclear transport receptors. The exact phase state of the permeability barrier remains elusive. In vitro experiments have shown that some FG-Nups can undergo phase separation into condensates that display NPC-like permeability barrier properties. Here, we use molecular dynamics simulations at amino acid resolution to study the phase separation characteristics of each of the disordered FG-Nups of the yeast NPC. We find that GLFG-Nups undergo phase separation and reveal that the FG motifs act as highly dynamic hydrophobic stickers that are essential for the formation of FG-Nup condensates featuring droplet-spanning percolated networks. Additionally, we study phase separation in an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple GLFG-Nups. We find that the phase separation of this NPC condensate is also driven by FG-FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed phase separation behavior, the different FG-Nups of the yeast NPC can be divided into two classes: The FG-Nups (mostly GLFG-type) located in the central channel of the NPC form a highly dynamic percolated network formed by many short-lived FG-FG interactions, while the peripheral FG-Nups (mostly FxFG-type) at the entry and exit of the NPC channel likely form an entropic brush.
... In the nuclear envelope (NE), NPCs are the stationary component of nuclear transport, mediating the mobile phase, comprising the bi-directional traffic of import of proteins to and export of RNAs from the nucleus [9][10][11]. Much of transport across the NPC is mediated by multiple members of the karyopherin (Kap) family of nuclear transport receptors (NTRs), at rates approaching 1000 molecules/NPC/sec ( [12,13] and references therein). ...
... Based on this information, a 'virtual gating' model was proposed in which dynamic multivalent interactions of NTRs with these FG repeats would provide just sufficient avidity to allow their rapid passage across the CT by overcoming entropic repulsion effects of the same IDR regions that otherwise exclude the passage of non-binding, non-specific macromolecules [22,27]. Indeed, it now seems evident that the mechanism for facilitated transport in the CT must include three features: first, the rate of facilitated transport across the NPC is similar to free diffusion within cells [28], so the internal mechanism of facilitated selection must be extraordinarily rapid; second, to maintain facilitated selection, the ratio of concentration of NTRs/cargoes to passive molecules (non-NTRs) within the CT must exceed that external to the CT, i, e, NTRs/cargoes must be relatively concentrated in the CT by interaction with it [9,[29][30][31]; and third, to be consistent with the first and second points, a simple mechanism of inhibition of non-NTR transport is entropic exclusion [10,27,32,33], although exactly how that mechanism plays out in the NPC is still unclear (below). We will now address how these features are produced by the NPC. ...
Nuclear pore complexes (NPCs) mediate the exchange of materials between the nucleoplasm and cytoplasm, playing a key role in the separation of nucleic acids and proteins into their required compartments. The static structure of the NPC is relatively well defined by recent cryo-EM and other studies. The functional roles of dynamic components in the pore of the NPC, phenylalanyl-glycyl (FG) repeat rich nucleoporins, is less clear because of our limited understanding of highly dynamic protein systems. These proteins form a 'restrained concentrate' which interacts with and concentrates nuclear transport factors (NTRs) to provide facilitated nucleocytoplasmic transport of cargoes. Very rapid on- and off-rates among FG repeats and NTRs supports extremely fast facilitated transport, close to the rate of macromolecular diffusion in cytoplasm, while complexes without specific interactions are entropically excluded, though details on several aspects of the transport mechanism and FG repeat behaviors remain to be resolved. However, as discussed here, new technical approaches combined with more advanced modeling methods will likely provide an improved dynamic description of NPC transport, potentially at the atomic level in the near future. Such advances are likely to be of major benefit in comprehending the roles the malfunctioning NPC plays in cancer, ageing, viral diseases, and neurodegeneration.
... In the nuclear envelope (NE), NPCs have two critical properties. First, they are the stationary phase of nuclear transport, mediating the mobile phase, comprising the bi-directional traffic of import of proteins to and export of RNAs from the nucleus (10,11). ...
... Based on this information, a "virtual gating" model was proposed in which dynamic multivalent interactions of NTRs with these FG repeats would provide sufficient avidity to allow their rapid passage across the CT by overcoming entropic repulsion effects of the same IDR regions that otherwise exclude the passage of non-binding, non-specific macromolecules (22,27). Indeed, it now seems evident that the mechanism for facilitated transport in the CT must include three features: first, the rate of facilitated transport across the NPC is similar to free diffusion within cells (28), so the internal mechanism of facilitated selection must be extraordinarily rapid; second, to maintain facilitated selection, the ratio of concentration of NTRs/cargoes to passive molecules (non-NTRs) within the CT must exceed that external to the CT, i, e, NTRs/cargoes must be relatively concentrated in the CT by interaction with it (9,(29)(30)(31); and third, to be consistent with the first and second points, the simplest mechanism of inhibition of non-NTR transport is entropic exclusion (10,27,32,33), although exactly how that mechanism plays out in the NPC is still unclear (below). We will now address how these features are produced by the NPC. ...
Nuclear pore complexes (NPCs) mediate the exchange of materials between the nucleoplasm and cytoplasm, playing a key role in the separation of nucleic acids and proteins into their required compartments. The static structure of the NPC is relatively well defined by recent cryo EM and other studies. The functional roles of dynamic components in the pore of the NPC, phenylalanyl-glycyl (FG) repeat rich nucleoporins, is less clear because of our limited understanding of highly dynamic protein systems. These proteins form a restrained concentrate which interacts with and concentrates nuclear transport factors (NTRs) to provide facilitated nucleocytoplasmic transport of cargoes. Very rapid exchange among FG repeats and NTRs supports extremely fast facilitated transport, close to the rate of macromolecular diffusion in cytoplasm, while complexes without specific interactions are entropically excluded, though details on several aspects of the transport mechanism and FG repeat behaviors remain to be resolved. However, as discussed here, new technical approaches combined with more advanced modeling methods will likely provide an improved dynamic description of NPC transport, potentially at the atomic level in the near future. Such advances are likely to be of major benefit in comprehending the roles the malfunctioning NPC plays in cancer, aging, viral diseases, and neurodegeneration.
... In the nuclear envelope (NE), NPCs have two critical properties. First, they are the stationary phase of nuclear transport, mediating the mobile phase, comprising the bi-directional traffic of import of proteins to and export of RNAs from the nucleus (10,11). ...
... Based on this information, a "virtual gating" model was proposed in which dynamic multivalent interactions of NTRs with these FG repeats would provide sufficient avidity to allow their rapid passage across the CT by overcoming entropic repulsion effects of the same IDR regions that otherwise exclude the passage of non-binding, non-specific macromolecules (22,27). Indeed, it now seems evident that the mechanism for facilitated transport in the CT must include three features: first, the rate of facilitated transport across the NPC is similar to free diffusion within cells (28), so the internal mechanism of facilitated selection must be extraordinarily rapid; second, to maintain facilitated selection, the ratio of concentration of NTRs/cargoes to passive molecules (non-NTRs) within the CT must exceed that external to the CT, i, e, NTRs/cargoes must be relatively concentrated in the CT by interaction with it (9,(29)(30)(31); and third, to be consistent with the first and second points, the simplest mechanism of inhibition of non-NTR transport is entropic exclusion (10,27,32,33), although exactly how that mechanism plays out in the NPC is still unclear (below). We will now address how these features are produced by the NPC. ...
Nuclear pore complexes (NPCs) mediate the exchange of materials between the nucleoplasm and cytoplasm, playing a key role in the separation of nucleic acids and proteins into their required compartments. The static structure of the NPC is relatively well defined by recent cryo EM and other studies. The functional roles of dynamic components in the pore of the NPC, phenylalanyl-glycyl (FG) repeat rich nucleoporins, is less clear because of our limited understanding of highly dynamic protein systems. These proteins form a restrained concentrate which interacts with and concentrates nuclear transport factors (NTRs) to provide facilitated nucleocytoplasmic transport of cargoes. Very rapid exchange among FG repeats and NTRs supports extremely fast facilitated transport, close to the rate of macromolecular diffusion in cytoplasm, while complexes without specific interactions are entropically excluded, though details on several aspects of the transport mechanism and FG repeat behaviors remain to be resolved. However, as discussed here, new technical approaches combined with more advanced modeling methods will likely provide an improved dynamic description of NPC transport, potentially at the atomic level in the near future. Such advances are likely to be of major benefit in comprehending the roles the malfunctioning NPC plays in cancer, aging, viral diseases, and neurodegeneration.
... First, in the RNA transport pathway, 2 genes YALI0_ C17567g and YALI0_D20658g were significantly upregulated by2.56-log2 and 1.30-log2 fold in YlΔA1-FAR strain, respectively. Among them, YALI0_C17567g gene encodes the nuclear pore complex (NPC) protein, which mediates the export of RNAs produced in the nucleus to fulfill their function in protein synthesis (Rout et al., 2000;Rout and Aitchison, 2001;Rodriguez et al., 2004). Therefore, the upregulation of YALI0_C17567g gene can facilitate the transport of RNAs out of the nucleus (Figure 5A). ...
Non-homologous end joining (NHEJ)-mediated integration is effective in generating random mutagenesis to identify beneficial gene targets in the whole genome, which can significantly promote the performance of the strains. Here, a novel target leading to higher protein synthesis was identified by NHEJ-mediated integration that seriously improved fatty alcohols biosynthesis in Yarrowia lipolytica. One batch of strains transformed with fatty acyl-CoA reductase gene (FAR) showed significant differences (up to 70.53-fold) in fatty alcohol production. Whole-genome sequencing of the high-yield strain demonstrated that a new target YALI0_A00913g (“A1 gene”) was disrupted by NHEJ-mediated integration of partial carrier DNA, and reverse engineering of the A1 gene disruption (YlΔA1-FAR) recovered the fatty alcohol overproduction phenotype. Transcriptome analysis of YlΔA1-FAR strain revealed A1 disruption led to strengthened protein synthesis process that was confirmed by sfGFP gene expression, which may account for enhanced cell viability and improved biosynthesis of fatty alcohols. This study identified a novel target that facilitated synthesis capacity and provided new insights into unlocking biosynthetic potential for future genetic engineering in Y. lipolytica.
... There are two subdomains: The nuclear envelope (NE) and the peripheral ER. Two flat membrane bilayers form the inner and outer nuclear membrane (INM/ONM) which serve as barrier between nuclear and cytoplasmic space [42,43]. Maintenance of the NE requires several interactions such as INM-associated protein binding to chromatin and lamina, linker proteins between INM and ONM for constant spacing, anchoring to cytoskeletal elements, and the positioning of nuclear pore complexes (NPCs) [41,44]. ...
Pathogenic intracellular bacteria, parasites and viruses have evolved sophisticated mechanisms to manipulate mammalian host cells to serve as niches for persistence and proliferation. The intracellular lifestyles of pathogens involve the manipulation of membrane-bound organellar compartments of host cells. In this review, we described how normal structural organization and cellular functions of endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, or lipid droplets are targeted by microbial virulence mechanisms. We focus on the specific interactions of Salmonella, Legionella pneumophila, Rickettsia rickettsii, Chlamydia spp. and Mycobacterium tuberculosis representing intracellular bacterial pathogens, and of Plasmodium spp. and Toxoplasma gondii representing intracellular parasites. The replication strategies of various viruses, i.e., Influenza A virus, Poliovirus, Brome mosaic virus, Epstein-Barr Virus, Hepatitis C virus, severe acute respiratory syndrome virus (SARS), Dengue virus, Zika virus, and others are presented with focus on the specific manipulation of the organelle compartments. We compare the specific features of intracellular lifestyle and replication cycles, and highlight the communalities in mechanisms of manipulation deployed.
... KPNA3 and KPNA4, both belonged to the karyopherin alpha two subfamily [13,14], play vital roles in protein nuclear transportation [15][16][17][18], nuclear pore complexes (NPC) assembly [19][20][21], and in regulating transcriptional activity [22,23], innate immune response [24][25][26] and cell death [27][28][29][30]. Previous studies also demonstrated that some viruses such as Japanese encephalitis virus [31], porcine circovirus type 2 [32], Middle East respiratory syndrome coronavirus [33] and influenza virus [34][35][36][37] could hijack KPNA3/4 to competitively block the interaction of KPNA3/4 with their cargos, and then to restrict their function to establish infection or evade the host antiviral response. ...
... This study was supported by Jiangsu Agricultural Science and Technology Innovation Fund [CX (19) ...
The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) have caused a huge economic loss to the poultry industry globally since 2013. Although the Fiber-2 has been identified as a key virulent related factor for FAdV-4, little is known about its molecular basis. In this study, we identified the efficient interaction of the Fiber-2 with the karyopherin alpha 3/4 (KPNA3/4) protein via its N-terminus of 1–40aa. The analysis of the overexpression and knockout of KPNA3/4 showed that KPNA3/4 could efficiently assist the replication of FAdV-4. Moreover, a fiber-2-edited virus FAV-4_Del with a deletion of 7–40aa in Fiber-2 was rescued through the CRISPR-Cas9 technique. In comparison with the wild type FAdV-4, FAV-4_Del was highly attenuated in vitro and in vivo. Notably, the inoculation of FAV-4_Del in chickens could provide full protection against the lethal challenge with the wild type FAdV-4. All these findings not only give novel insights into the molecular basis for the pathogenesis of Fiber-2 but also provide efficient targets for developing antiviral strategies and live-attenuated vaccine candidates against the highly pathogenic FAdV-4.
... [6][7][8] Second, they provide binding sites for nuclear transport receptors (NTRs), 9 that rapidly ferry cargo molecules through the NPC, with energy and directionality contributed by the Ran GTPase. 10 A mechanistic understanding of the NPC's gatekeeping function is not only essential to the study of cellular compartmentalization, but also provides design inspirations for synthetic macromolecule-sorting machines. ...
DNA nanotechnology provides a versatile and powerful tool to dissect the structure-function relationship of biomolecular machines like the nuclear pore complex (NPC), an enormous protein assembly that controls molecular traffic between the nucleus and cytoplasm. To understand how the intrinsically disordered, Phe-Gly-rich nucleoporins (FG-nups) within the NPC's central transport channel impede the diffusion of macromolecules, we built a DNA-origami NanoTrap. The NanoTrap comprises precisely arranged FG-nups in an NPC-like channel, which sits on a baseplate that captures macromolecules that pass through the FG network. Using this biomimetic construct, we determined that the FG-motif type, grafting density and spatial arrangement are critical determinants of an effective diffusion barrier. Further, we observe that diffusion barriers formed with cohesive FG-interactions dominate in mixed-FG-nup scenarios. Our DNA-origami platform thus sheds light on how NPCs sieve inert macromolecules and will provide a valuable tool for studying nuclear transport.
... Of course, phosphatases play a similar role in signal transduction but they are a less intensively studied area compared to kinases [64]. Proteins travel into and out of the cell nucleus exclusively through the nuclear pore complex, which is an elaborate constellation of almost 50 distinct components embedded in the nuclear envelope [65]. Small proteins of less than 50 kD s diffuse freely into and out of the nucleus noting that most common signalling molecules are about this size or smaller [65]. ...
... Proteins travel into and out of the cell nucleus exclusively through the nuclear pore complex, which is an elaborate constellation of almost 50 distinct components embedded in the nuclear envelope [65]. Small proteins of less than 50 kD s diffuse freely into and out of the nucleus noting that most common signalling molecules are about this size or smaller [65]. Two major factors that affect protein partitioning between the cytosol and the nucleus are interactions with the nuclear transport machinery and interactions with anchoring proteins which reside stably in the nucleus or the cytosol. ...
This review will cover the signalling pathways leading to the phosphorylation of the Smad linker region independent of Smad carboxy terminal phosphorylation. Characterising Smad linker region as a signalling pathway in its own right will encourage comprehensive signalling studies to provide solutions for successful discovery and exploitation of drug targets. The review describes Smad transcription factor signalling distinct from Transforming Growth Factor (TGF)-β signalling. Novel signalling pathways represent new drug targets where these pathways are known to be involved in fibrosis, cancer and cardiovascular disease.
