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Background Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture. Objectives As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites). Methods Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested. Results Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation. Conclusions As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs. [ABSTRACT FROM AUTHOR]

Abstract Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and facilitate the exchange of macromolecules between the nucleus and cytoplasm in eukaryotic cells. The dysfunction of the NPC and nuclear transport plays a significant role in aging and the pathogenesis of various neurodegenerative diseases. Common features among these neurodegenerative diseases, including Parkinson’s disease (PD), encompass mitochondrial dysfunction, oxidative stress and the accumulation of insoluble protein aggregates in specific brain regions. The susceptibility of dopaminergic neurons to mitochondrial stress underscores the pivotal role of mitochondria in PD progression. Disruptions in mitochondrial-nuclear communication are exacerbated by aging and α-synuclein-induced oxidative stress in PD. The precise mechanisms underlying mitochondrial impairment-induced neurodegeneration in PD are still unclear. Evidence suggests that perturbations in dopaminergic neuronal nuclei are linked to PD-related neurodegeneration. These perturbations involve structural damage to the nuclear envelope and mislocalization of pivotal transcription factors, potentially driven by oxidative stress or α-synuclein pathology. The presence of protein aggregates, pathogenic mutations, and ongoing oxidative stress can exacerbate the dysfunction of NPCs, yet this mechanism remains understudied in the context of oxidative stress-induced PD. This review summarizes the link between mitochondrial dysfunction and dopaminergic neurodegeneration and outlines the current evidence for nuclear envelope and nuclear transport abnormalities in PD, particularly in oxidative stress. We highlight the potential role of nuclear pore and nucleocytoplasmic transport dysfunction in PD and stress the importance of systematically investigating NPC components in PD.

SUMMARY Proteins in the importin α (IMPA) family play pivotal roles in intracellular nucleocytoplasmic transport. Arabidopsis thaliana possesses nine IMPA members, with diverse tissue‐specific expression patterns. Among these nine IMPAs, IMPA1, IMPA2, and IMPA4 cluster together phylogenetically, suggesting potential functional redundancy. To explore this redundancy, we analyzed single and multiple T‐DNA mutants for these genes and discovered severe growth defects in the impa1 impa2 impa4 triple knockout mutant but not in the single or double mutants. Complementation with IMPA1, IMPA2, or IMPA4 fused to green fluorescent protein (GFP) rescued the growth defects observed in the impa1 impa2 impa4 mutant, indicating the functional redundancy of these three IMPAs. The IMPA‐GFP fusion proteins were localized in the nucleus and nuclear envelope, suggesting their involvement in nucleocytoplasmic transport processes. Comparative transcriptomics revealed that salicylic acid (SA)‐responsive genes were significantly upregulated in the impa1 impa2 impa4 triple mutant. Consistent with this observation, impa1 impa2 impa4 mutant plants accumulated SA and reactive oxygen species to high levels compared with wild‐type plants. We also found enhanced resistance to the anthracnose pathogen Colletotrichum higginsianum in the impa1 impa2 impa4 mutants, suggesting that defense responses were constitutively activated in the impa1 impa2 impa4 mutant. Our findings shed light on the redundant roles of IMPA1, IMPA2, and IMPA4 in suppressing the autoimmune responses and suggest avenues of research to clarify their potentially unique roles. [ABSTRACT FROM AUTHOR]

The analysis of nucleocytoplasmic transport of proteins and messenger RNA has been the focus of advanced microscopic approaches. Recently, it has been possible to identify and visualize individual pre-ribosomal particles on their way through the nuclear pore complex using both electron and light microscopy. In this review, we focused on the transport of pre-ribosomal particles in the nucleus on their way to and through the pores. [ABSTRACT FROM AUTHOR]

Cell division presents a challenge for eukaryotic cells: how can chromosomes effectively segregate within the confines of a membranous nuclear compartment? Different organisms have evolved diverse solutions by modulating the degree of nuclear compartmentalization, ranging from complete nuclear envelope breakdown to complete maintenance of nuclear compartmentalization via nuclear envelope expansion. Many intermediate forms exist between these extremes, suggesting that nuclear dynamics during cell division are surprisingly plastic. In this review, we highlight the evolutionary diversity of nuclear divisions, focusing on two defining characteristics: (1) chromosome compartmentalization and (2) nucleocytoplasmic transport. Further, we highlight recent evidence that nuclear behavior during division can vary within different cellular contexts in the same organism. The variation observed within and between organisms underscores the dynamic evolution of nuclear divisions tailored to specific contexts and cellular requirements. In-depth investigation of diverse nuclear divisions will enhance our understanding of the nucleus, both in physiological and pathological states. [ABSTRACT FROM AUTHOR]

The separation of genetic material from bulk cytoplasm has enabled the evolution of increasingly complex organisms, allowing for the development of sophisticated forms of life. However, this complexity has created new categories of dysfunction, including those related to the movement of material between cellular compartments. In eukaryotic cells, nucleocytoplasmic trafficking is a fundamental biological process, and cumulative disruptions to nuclear integrity and nucleocytoplasmic transport are detrimental to cell survival. This is particularly true in post-mitotic neurons, where nuclear pore injury and errors to nucleocytoplasmic trafficking are strongly associated with neurodegenerative disease. In this review, we summarize the current understanding of nuclear pore biology in physiological and pathological contexts and discuss potential therapeutic approaches for addressing nuclear pore injury and dysfunctional nucleocytoplasmic transport. [ABSTRACT FROM AUTHOR]

Splicing factor proline‐glutamine rich (SFPQ), a multifunctional RNA‐binding protein (RBP), shows cytoplasmic colocalisation with stress granule (SG) markers; however, the causative relationship and mechanism underlying this coalescence of SFPQ aggregates and SGs remain unclear. In this study, we demonstrate that SFPQ lacking its nuclear localisation sequence spontaneously forms cytoplasmic aggregates that abnormally incorporate immature RNA and induce persistent SGs. mRNA profiling showed that SFPQ mislocalisation induced extensive changes in RNA processing, with a subset of alternatively spliced transcripts associated with nucleocytoplasmic transport. Notably, these altered transporters were sequestered into SFPQ aggregates, constituting aberrant protein‐RNA complexes. Importantly, suppression of SG nucleation could not block cytoplasmic SFPQ aggregation with immature RNA and nucleocytoplasmic transporters, both of which, however, were moderately ameliorated by the inhibition of alternative splicing or nuclear export. Our results unveil the physiopathological role and mechanism for mislocalised SFPQ in the RNA metabolism, nucleocytoplasmic transport and pathological SGs. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and facilitate the exchange of macromolecules between the nucleus and cytoplasm in eukaryotic cells. The dysfunction of the NPC and nuclear transport plays a significant role in aging and the pathogenesis of various neurodegenerative diseases. Common features among these neurodegenerative diseases, including Parkinson's disease (PD), encompass mitochondrial dysfunction, oxidative stress and the accumulation of insoluble protein aggregates in specific brain regions. The susceptibility of dopaminergic neurons to mitochondrial stress underscores the pivotal role of mitochondria in PD progression. Disruptions in mitochondrial-nuclear communication are exacerbated by aging and α-synuclein-induced oxidative stress in PD. The precise mechanisms underlying mitochondrial impairment-induced neurodegeneration in PD are still unclear. Evidence suggests that perturbations in dopaminergic neuronal nuclei are linked to PD-related neurodegeneration. These perturbations involve structural damage to the nuclear envelope and mislocalization of pivotal transcription factors, potentially driven by oxidative stress or α-synuclein pathology. The presence of protein aggregates, pathogenic mutations, and ongoing oxidative stress can exacerbate the dysfunction of NPCs, yet this mechanism remains understudied in the context of oxidative stress-induced PD. This review summarizes the link between mitochondrial dysfunction and dopaminergic neurodegeneration and outlines the current evidence for nuclear envelope and nuclear transport abnormalities in PD, particularly in oxidative stress. We highlight the potential role of nuclear pore and nucleocytoplasmic transport dysfunction in PD and stress the importance of systematically investigating NPC components in PD. [ABSTRACT FROM AUTHOR]

Background/Objectives: Changes in nuclear morphology, alterations to the nuclear pore complex (NPC), including loss, aggregation, and dysfunction of nucleoporins (Nups), and nucleocytoplasmic transport (NCT) abnormalities have become hallmarks of neurodegenerative diseases. Previous RNA sequencing data utilizing knockdown of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) identified enrichment for pathways and changes in RNAs related to nuclear morphology and showed differential expression of key nuclear targets. This suggests that dysfunction of hnRNP A1, which is observed in neurodegenerative diseases, may contribute to abnormalities in nuclear morphology, NPC, and NCT. Methods: We performed knockdown of hnRNP A1 in Neuro-2A cells, a neuronal cell line, to examine nuclear morphology, NPC, and NCT. Results: First, we examined nuclear morphology using Lamin B, wherein we observed increased nuclear envelope abnormalities in cells with hnRNP A1 knockdown as compared to control. To quantify changes in Lamin B, we designed and validated an automated computer-based model, which quantitatively confirmed our observations. Next, we investigated the impact of hnRNP A1 knockdown on components of the NPC and NCT. In line with the previous literature, we found changes in Nups, including altered distribution and reduced protein expression, as well as disrupted NCT. Finally, we validated our findings in multiple sclerosis (MS) brains, a disease with a significant neurodegenerative component caused by hnRNP A1 dysfunction, where neuronal nuclear envelope alterations were significantly increased as compared to controls. Conclusions: Together, these data implicate hnRNP A1 as an important contributor to nuclear morphology, Nup expression and distribution, and NCT and suggest that hnRNP A1 dysfunction may lead to defects in these processes in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

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.

Heat stress strongly triggers the nuclear localization of the molecular chaperone HSP70. Hikeshi functions as a unique nuclear import carrier of HSP70. However, how the nuclear import of HSP70 is activated in response to heat stress remains unclear. Here, we investigated the effects of heat on the nuclear import of HSP70. In vitro transport assays revealed that pretreatment of the test samples with heat facilitated the nuclear import of HSP70. Furthermore, binding of Hikeshi to HSP70 increased when temperatures rose. These results indicated that heat is one of the factors that activates the nuclear import of HSP70. Previous studies showed that the F97A mutation in Hikeshi in an extended loop induced an opening in the hydrophobic pocket and facilitated the translocation of Hikeshi through the nuclear pore complex. We found that nuclear accumulation of HSP70 occurred at a lower temperature in cells expressing the Hikeshi‐F97A mutant than in cells expressing wild‐type Hikeshi. Collectively, our results show that the movement of the extended loop may play an important role in the interaction of Hikeshi with both FG (phenylalanine‐glycine)‐nucleoporins and HSP70 in a temperature‐dependent manner, resulting in the activation of nuclear import of HSP70 in response to heat stress. [ABSTRACT FROM AUTHOR]

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease. Synopsis: Nuclear exclusion of TDP-43 is observed in various pathologies, but the physiological mechanisms that ensure its nuclear localization are not well-known. This work shows that inhibition of persistent Wnt activation in neurons by GDE2 prevents TDP-43 nuclear exclusion. GDE2 inhibits canonical Wnt signaling in adult postmitotic neurons. Sustained activation of canonical Wnt signaling in neurons disrupts the nuclear pore complex, impairs nucleocytoplasmic transport, and results in TDP-43 nuclear exclusion. iPS neurons from patients with C9orf72 ALS show decreased GDE2 expression and increased activation of canonical Wnt signaling. Inhibition of Wnt activation mitigates TDP-43 dysfunction in C9orf72 iPS neurons. GDE2 maintains TDP-43 nuclear localization by inhibiting Wnt activation in neurons. [ABSTRACT FROM AUTHOR]

Premature ovarian insufficiency (POI) is defined as the depletion of ovarian function before the age of 40 years. The global prevalence of POI is 3.5%. To date, genetic factors account for 23.5% of the etiology of POI. Herein, a previously uncharacterized pathogenic homozygous variant of the chromosome segregation–1–like gene (CSE1L) was identified in POI patients via targeted panel sequencing. It is reported that dysregulated iron metabolism is involved in many reproductive endocrine disorders; however, its precise role in POI remains obscure. In this study, we identified CSE1L as a potential candidate gene that plays an important role in maintaining iron homeostasis. Deficiency of CSE1L led to ferroptosis in human granulosa cells, which was confirmed by transmission electron microscopy. Mechanistically, coimmunoprecipitation identified the direct interaction between CSE1L and FoxO1. Inhibition of CSE1L led to the excessive accumulation of FoxO1 in the nucleus via nucleocytoplasmic transport. Then, FoxO1 bound to the promoter region of NCOA4 and promoted its transcription, which was verified by a chromatin immunoprecipitation assay. Moreover, inhibition of CSE1L in cumulus cell monolayer could impede oocyte maturation, which might be associated with oxidative stress. Consequently, our study first revealed that CSE1L participated in ferroptosis in human ovarian granulosa cells via nucleocytoplasmic transportation, which might be helpful in revealing the molecular mechanism of CSE1L in the development of POI. Importantly, these findings might provide new insights into the application of ferroptosis inhibitors in the treatment of POI. [ABSTRACT FROM AUTHOR]

Expansion of the GGGGCC-RNA repeat is a known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which currently have no cure. Recent studies have indicated the activation of Sigma-1 receptor plays an important role in providing neuroprotection, especially in ALS and Alzheimer's disease. Nevertheless, the mechanisms underlying Sigma-1R activation and its effect on (G4C2)n-RNA-induced cell death remain unclear. In this study, we demonstrated that fluvoxamine is a Sigma-1R agonist that can increase chaperone activity and stabilize the protein expression of Pom121 in (G4C2)31-RNA-expressing NSC34 cells, leading to increased colocalization at the nuclear envelope. Interestingly, fluvoxamine treatment increased Pom121 protein expression without affecting transcription. In C9orf72-ALS, the nuclear translocation of TFEB autophagy factor decreased owing to nucleocytoplasmic transport defects. Our results showed that pretreatment of NSC34 cells with fluvoxamine promoted the shuttling of TFEB into the nucleus and elevated the expression of LC3-II compared to the overexpression of (G4C2)31-RNA alone. Additionally, even when used alone, fluvoxamine increases Pom121 expression and TFEB translocation. To summarize, fluvoxamine may act as a promising repurposed medicine for patients with C9orf72-ALS, as it stabilizes the nucleoporin Pom121 and promotes the translocation of TFEB in (G4C2)31-RNA-expressing NSC34 cells. [ABSTRACT FROM AUTHOR]

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patientspecific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RANbinding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Different neural subtypes are selectively lost in diverse neurodegenerative diseases. Huntington’s disease (HD) is an inherited neurodegenerative disease characterized by motor abnormalities that primarily affect the striatum. The Huntingtin (HTT) mutation involves an expanded CAG repeat, leading to insoluble polyQ, which renders GABA+ medium spiny neurons (MSN) more venerable to cell death. Human pluripotent stem cells (hPSCs) technology allows for the construction of disease-specific models, providing valuable cellular models for studying pathogenesis, drug screening, and high-throughput analysis. Methods In this study, we established a method that allows for rapid and efficient generation of MSNs (> 90%) within 21 days from hPSC-derived neural progenitor cells, by introducing a specific combination of transcription factors. Results We efficiently induced several neural subtypes, in parallel, based on the same cell source, and revealed that, compared to other neural subtypes, MSNs exhibited higher polyQ aggregation propensity and overexpression toxicity, more severe dysfunction in BDNF/TrkB signaling, greater susceptibility to BDNF withdrawal, and more severe disturbances in nucleocytoplasmic transport (NCT). We further found that the nuclear lamina protein LMNB1 was greatly reduced in HD neurons and mislocalized to the cytoplasm and axons. Knockdown of HTT or treatment with KPT335, an orally selective inhibitor of nuclear export (SINE), effectively attenuated the pathological phenotypes and alleviated neuronal death caused by BDNF withdrawal. Conclusions This study thus establishes an effective method for obtaining MSNs and underscores the necessity of using high-purity MSNs to study HD pathogenesis, especially the MSN-selective vulnerability.

Abstract Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the cytoplasmic mislocalization and aberrant phase transitions of prion-like RNA-binding proteins (RBPs). The common accumulation of TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), and other nuclear RBPs in detergent-insoluble aggregates in the cytoplasm of degenerating neurons in ALS/FTD is connected to nuclear pore dysfunction and other defects in the nucleocytoplasmic transport machinery. Recent advances suggest that beyond their canonical role in the nuclear import of protein cargoes, nuclear-import receptors (NIRs) can prevent and reverse aberrant phase transitions of TDP-43, FUS, and related prion-like RBPs and restore their nuclear localization and function. Here, we showcase the NIR family and how they recognize cargo, drive nuclear import, and chaperone prion-like RBPs linked to ALS/FTD. We also discuss the promise of enhancing NIR levels and developing potentiated NIR variants as therapeutic strategies for ALS/FTD and related neurodegenerative proteinopathies. Graphical Abstract

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.

The small compound Pitstop‐2 is a recent potent inhibitor of clathrin‐mediated endocytosis (CME), widely used in biomedical research areas. In recent years, however, it is observed that it exhibits CME‐independent inhibitory effects on nuclear pore complexes (NPCs), the nucleocytoplasmic gatekeepers. NPCs are elaborate proteinaceous transport nano‐machineries of crucial physiological importance rendering them novel targets for various medical applications. They mediate all nucleocytoplasmic transport forming a physiologically essential selective nucleocytoplasmic barrier. The direct Pitstop‐2 disruptive effects on NPCs manifested themselves at both the structural and functional integrity levels. Moreover, they are massive, acute, and detectable at concentrations equal to CME‐inhibitory concentrations. Pitstop‐2 inhibits CME by binding to the terminal β‐propeller domain of the heavy chain of clathrin. Several NPC scaffold proteins, critical for the structural and functional integrity of the NPC, possess β‐propeller folds. Herein, utilizing computational docking analysis, it is demonstrated that Pitstop‐2 exhibits particularly high binding affinities to β‐propeller folds of NPC scaffold proteins, similar to its binding affinity to the terminal β‐propeller domain of clathrin. The authors, therefore, conclude that Pitstop‐2 is a potent disruptor of NPCs, an activity which, separately or in synergy with CME inhibition, may be exploited for a myriad of pharmacological applications. [ABSTRACT FROM AUTHOR]

Main conclusion: Physiological and molecular tests show that NUP96 plays an important role in the plant response to salt stress, resulting from the reprogramming of transcriptomic profiles, which are likely to be mediated by the influence on the nuclear/cytosol shuttling of the key regulators of salt tolerance. As a key component of the nuclear pore complex (NPC), nucleoporin 96 (NUP96) is critical for modulating plant development and interactions with environmental factors, but whether NUP96 is involved in the salt response is still unknown. Here, we analyzed the role of Arabidopsis NUP96 under salt stress. The loss-of-function mutant nup96 exhibited salt sensitivity in terms of rosette growth and root elongation, and showed attenuated capacity in maintaining ion and ROS homeostasis, which could be compensated for by the overexpression of NUP96. RNA sequencing revealed that many salt-responsive genes were misregulated after NUP96 mutation, and especially NUP96 is required for the expression of a large portion of salt-induced genes. This is likely correlated with the activity in facilitating nuclear/cytosol transport of the underlying regulators in salt tolerance such as the transcription factor ATAP2, targeted by eight downregulated genes in nup96 under salt stress. Our results illustrate that NUP96 plays an important role in the salt response, probably by regulating the nucleocytoplasmic shuttling of key mRNAs or proteins associated with plant salt responsiveness. [ABSTRACT FROM AUTHOR]

Nuclear import and uncoating of the viral capsid are critical steps in the HIV-1 life cycle that serve to transport and release genomic material into the nucleus. Viral core import involves translocating the HIV-1 capsid at the nuclear pore complex (NPC). Notably, the central channel of the NPC appears to often accommodate and allow passage of intact HIV-1 capsid, though mechanistic details of the process remain to be fully understood. Here, we investigate the molecular interactions that operate in concert between the HIV-1 capsid and the NPC that regulate capsid translocation through the central channel. To this end, we develop a "bottom-up" coarse-grained (CG) model of the human NPC from recently released cryo-electron tomography structure and then construct composite membrane-embedded CG NPC models. We find that successful translocation from the cytoplasmic side to the NPC central channel is contingent on the compatibility of the capsid morphology and channel dimension and the proper orientation of the capsid approach to the channel from the cytoplasmic side. The translocation dynamics is driven by maximizing the contacts between phenylalanine-glycine nucleoporins at the central channel and the capsid. For the docked intact capsids, structural analysis reveals correlated striated patterns of lattice disorder likely related to the intrinsic capsid elasticity. Uncondensed genomic material inside the docked capsid augments the overall lattice disorder of the capsid. Our results suggest that the intrinsic "elasticity" can also aid the capsid to adapt to the stress and remain structurally intact during translocation. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the cytoplasmic mislocalization and aberrant phase transitions of prion-like RNA-binding proteins (RBPs). The common accumulation of TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), and other nuclear RBPs in detergent-insoluble aggregates in the cytoplasm of degenerating neurons in ALS/FTD is connected to nuclear pore dysfunction and other defects in the nucleocytoplasmic transport machinery. Recent advances suggest that beyond their canonical role in the nuclear import of protein cargoes, nuclear-import receptors (NIRs) can prevent and reverse aberrant phase transitions of TDP-43, FUS, and related prion-like RBPs and restore their nuclear localization and function. Here, we showcase the NIR family and how they recognize cargo, drive nuclear import, and chaperone prion-like RBPs linked to ALS/FTD. We also discuss the promise of enhancing NIR levels and developing potentiated NIR variants as therapeutic strategies for ALS/FTD and related neurodegenerative proteinopathies. [ABSTRACT FROM AUTHOR]

Coordination and information exchange among the various organelles ensure the precise and orderly functioning of eukaryotic cells. Interaction between the cytoplasm and nucleoplasm is crucial for many physiological processes. Macromolecular protein transport into the nucleus requires assistance from the nuclear transport system. These proteins typically contain a nuclear localisation sequence that guides them to enter the nucleus. Understanding the mechanism of nuclear import of macromolecular proteins is important for comprehending cellular processes. Investigation of disease-related alterations can facilitate the development of novel therapeutic strategies and provide additional evidence for clinical trials. This review provides an overview of the proteins involved in nuclear transport and the mechanisms underlying macromolecular protein transport. [ABSTRACT FROM AUTHOR]

Persistent cytoplasmic aggregates containing RNA binding proteins (RBPs) are central to the pathogenesis of late-onset neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). These aggregates share components, molecular mechanisms, and cellular protein quality control pathways with stress-induced RNA granules (SGs). Here, we assess the impact of stress on the global mRNA localization landscape of human pluripotent stem cell-derived motor neurons (PSC-MNs) using subcellular fractionation with RNA sequencing and proteomics. Transient stress disrupts subcellular RNA and protein distributions, alters the RNA binding profile of SG- and ALS-relevant RBPs and recapitulates disease-associated molecular changes such as aberrant splicing of STMN2. Although neurotypical PSC-MNs re-establish a normal subcellular localization landscape upon recovery from stress, cells harboring ALS-linked mutations are intransigent and display a delayed-onset increase in neuronal cell death. Our results highlight subcellular molecular distributions as predictive features and underscore the utility of cellular stress as a paradigm to study ALS-relevant mechanisms.

Premature ovarian insufficiency (POI) is defined as the depletion of ovarian function before the age of 40 years. The global prevalence of POI is 3.5%. To date, genetic factors account for 23.5% of the etiology of POI. Herein, a previously uncharacterized pathogenic homozygous variant of the chromosome segregation–1–like gene (CSE1L) was identified in POI patients via targeted panel sequencing. It is reported that dysregulated iron metabolism is involved in many reproductive endocrine disorders; however, its precise role in POI remains obscure. In this study, we identified CSE1L as a potential candidate gene that plays an important role in maintaining iron homeostasis. Deficiency of CSE1L led to ferroptosis in human granulosa cells, which was confirmed by transmission electron microscopy. Mechanistically, coimmunoprecipitation identified the direct interaction between CSE1L and FoxO1. Inhibition of CSE1L led to the excessive accumulation of FoxO1 in the nucleus via nucleocytoplasmic transport. Then, FoxO1 bound to the promoter region of NCOA4 and promoted its transcription, which was verified by a chromatin immunoprecipitation assay. Moreover, inhibition of CSE1L in cumulus cell monolayer could impede oocyte maturation, which might be associated with oxidative stress. Consequently, our study first revealed that CSE1L participated in ferroptosis in human ovarian granulosa cells via nucleocytoplasmic transportation, which might be helpful in revealing the molecular mechanism of CSE1L in the development of POI. Importantly, these findings might provide new insights into the application of ferroptosis inhibitors in the treatment of POI.

The disruption of nucleocytoplasmic transport (NCT) is an important mechanism in neurodegenerative diseases. In the case of C9orf72-ALS, trafficking of macromolecules through the nuclear pore complex (NPC) might get frustrated by the binding of C9orf72-translated arginine-containing dipeptide repeat proteins (R-DPRs) to the Kapβ family of nuclear transport receptors. Besides Kapβs, several other types of transport components have been linked to NCT impairments in R-DPR-expressed cells, but the molecular origin of these observations has not been clarified. Here, we adopt a coarse-grained molecular dynamics model at amino acid resolution to study the direct interaction between polyPR, the most toxic DPR, and various nuclear transport components to elucidate the binding mechanisms and provide a complete picture of potential polyPR-mediated NCT defects. We found polyPR to directly bind to several isoforms of the Impα family, CAS (the specific exporter of Impα) and RanGAP. We observe no binding between polyPR and Ran. Longer polyPRs at lower salt concentrations also make contact with RanGEF and NTF2. Analyzing the polyPR contact sites on the transport components reveals that polyPR potentially interferes with RanGTP/RanGDP binding, with nuclear localization signal (NLS)-containing cargoes (cargo-NLS) binding to Impα, with cargo-NLS release from Impα, and with Impα export from the nucleus. The abundance of polyPR-binding sites on multiple transport components combined with the inherent polyPR length dependence makes direct polyPR interference of NCT a potential mechanistic pathway of C9orf72 toxicity.

ABSTRACT Viruses have evolved intricate mechanisms to evade host antiviral responses and exploit cellular resources by manipulating the expression profile of host genes. During infection, viruses encode proteins with shutoff activity to globally inhibit host protein synthesis, which is an effective strategy for immune evasion. In this study, compelling evidence shows that infectious bursal disease virus (IBDV) infection triggers the suppression of host protein synthesis. Furthermore, using both in vitro and in vivo viral infection models, we have identified that IBDV specifically impedes the transcription of host genes via the shutoff activity of viral VP5, simultaneously conferring advantages to IBDV infection in these circumstances. The proposed mechanism suggests that VP5 competitively binds to RanBP1, disrupting the RanGDP/GTP gradient. This disruption interferes with cellular nucleocytoplasmic transport, impairing the nuclear import of proteins bearing nuclear localization signals. The nuclear transport of pivotal transcriptional regulatory factors, such as p65 and IFN regulatory factor 7, is also compromised, leading to the inhibition of pro-inflammatory cytokines and interferon expression. This newly discovered strategy employed by IBDV enables them to manipulate host gene expression, providing novel insights into how viruses evade host immune responses and establish infections.IMPORTANCEViruses manipulate host processes at various levels to regulate or evade both innate and adaptive immune responses, promoting self-survival and efficient transmission. The “host shutoff,” a global suppression of host gene expression mediated by various viruses, is considered a critical mechanism for evading immunity. In this study, we have validated the presence of host shutoff during infectious bursal disease virus (IBDV) infection and additionally uncovered that the viral protein VP5 plays a pivotal role in inhibiting the overall synthesis of host proteins, including cytokines, through a transcription-dependent pathway. VP5 competitively binds with RanBP1, leading to disruption of the Ran protein cycle and consequently interfering with nucleocytoplasmic transport, which ultimately results in the suppression of host gene transcription. These findings unveil a novel strategy employed by IBDV to evade host innate immunity and rapidly establish infection. This study also suggests a novel supplement to understanding the pathway through which viruses inhibit host protein synthesis.

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

Ran‐binding protein 2 (RANBP2)/Nup358 is a nucleoporin and a key component of the nuclear pore complex. Through its multiple functions (e.g., SUMOylation, regulation of nucleocytoplasmic transport) and subcellular localizations (e.g., at the nuclear envelope, kinetochores, annulate lamellae), it is involved in many cellular processes. RANBP2 dysregulation or mutation leads to the development of human pathologies, such as acute necrotizing encephalopathy 1, cancer, neurodegenerative diseases, and it is also involved in viral infections. The chromosomal region containing the RANBP2 gene is highly dynamic, with high structural variation and recombination events that led to the appearance of a gene family called RANBP2 and GCC2 Protein Domains (RGPD), with multiple gene loss/duplication events during ape evolution. Although RGPD homoplasy and maintenance during evolution suggest they might confer an advantage to their hosts, their functions are still unknown and understudied. In this review, we discuss the appearance and importance of RANBP2 in metazoans and its function‐related pathologies, caused by an alteration of its expression levels (through promotor activity, post‐transcriptional, or post‐translational modifications), its localization, or genetic mutations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nucleocytoplasmic transport comprises the multistep assembly, transport, and disassembly of protein and RNA cargoes entering and exiting nuclear pores. Accruing evidence supports that impairments to nucleocytoplasmic transport are a hallmark of neurodegenerative diseases. These impairments cause dysregulations in nucleocytoplasmic partitioning and proteostasis of nuclear transport receptors and client substrates that promote intracellular deposits – another hallmark of neurodegeneration. Disturbances in liquid–liquid phase separation (LLPS) between dense and dilute phases of biomolecules implicated in nucleocytoplasmic transport promote micrometer‐scale coacervates, leading to proteinaceous aggregates. This Review provides historical and emerging principles of LLPS at the interface of nucleocytoplasmic transport, proteostasis, aging and noxious insults, whose dysregulations promote intracellular aggregates. E3 SUMO‐protein ligase Ranbp2 constitutes the cytoplasmic filaments of nuclear pores, where it acts as a molecular hub for rate‐limiting steps of nucleocytoplasmic transport. A vignette is provided on the roles of Ranbp2 in nucleocytoplasmic transport and at the intersection of proteostasis in the survival of photoreceptor and motor neurons under homeostatic and pathophysiological environments. Current unmet clinical needs are highlighted, including therapeutics aiming to manipulate aggregation‐dissolution models of purported neurotoxicity in neurodegeneration. [ABSTRACT FROM AUTHOR]

Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. Here, we identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.

The transport of proteins to and from the nucleus is necessary for many cellular processes and is one of the ways plants respond to developmental signals and environmental stresses. Nucleocytoplasmic trafficking of proteins is mediated by the nuclear transport receptor (NTR). Although NTR has been extensively studied in humans and Arabidopsis, it has rarely been identified and functionally characterized in rice. In this study, we identified exportin 1 in rice (OsXPO1) as a nuclear export receptor. OsXPO1 shares high protein identity with its functional homologs in Arabidopsis and other organisms. OsXPO1 localized to both the nucleus and the cytoplasm, directly interacted with the small GTPases OsRAN1 and OsRAN2 in the nucleus, and mediated their nuclear export. Loss-of-function osxpo1 mutations were lethal at the seedling stage. Suppression of OsXPO1 expression in RNA interference lines produced multifaceted developmental defects, including arrested growth, premature senescence, abnormal inflorescence, and brown and mouth-opened spikelets. Overexpression of OsXPO1 in rice reduced plant height and seed-setting rate, but increased plant tolerance in response to PEG-mimicked drought stress and salt stress. These results indicate that OsXPO1 is a nuclear export receptor and acts in regulating plant development and abiotic stress responses.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two adult-onset neurodegenerative diseases that are part of a common disease spectrum due to clinical, genetic, and pathological overlap. A prominent genetic factor contributing to both diseases is a hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene. This mutation in C9orf72 leads to nuclear depletion and cytoplasmic aggregation of Tar DNA-RNA binding protein 43 (TDP-43). TDP-43 pathology is characteristic of the majority of ALS cases, irrespective of disease causation, and is present in ~50% of FTD cases. Defects in nucleocytoplasmic transport involving the nuclear pore complex, the Ran-GTPase cycle, and nuclear transport factors have been linked with the mislocalization of TDP-43. Here, we will explore and discuss the implications of these system abnormalities of nucleocytoplasmic transport in C9orf72-ALS/FTD, as well as in other forms of familial and sporadic ALS. [ABSTRACT FROM AUTHOR]

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.

One of the key features of eukaryotic cells is the separation of nuclear and cytoplasmic compartments by a double-layer nuclear envelope. This separation is crucial for timely regulation of gene expression, mRNA biogenesis, cell cycle, and differentiation. Since transcription takes place in the nucleus and the major part of translation in the cytoplasm, proper distribution of biomolecules between these two compartments is ensured by nucleocytoplasmic shuttling proteins – karyopherins. Karyopherins transport biomolecules through nuclear pores bidirectionally in collaboration with Ran GTPases and utilize GTP as the source of energy. Different karyopherins transport different cargo molecules that play important roles in the regulation of cell physiology. In cancer cells, this nucleocytoplasmic transport is significantly dysregulated to support increased demands for the import of cell cycle-promoting biomolecules and export of cell cycle inhibitors and mRNAs. Here, we analysed genomic, transcriptomic and proteomic data from published datasets to comprehensively profile karyopherin genes in hepatocellular carcinoma. We have found out that expression of multiple karyopherin genes is increased in hepatocellular carcinoma in comparison to the normal liver, with importin subunit α-1, exportin 2, importin subunit β-1 and importin 9 being the most over-expressed. Moreover, we have found that increased expression of these genes is associated with higher neoplasm grade as well as significantly worse overall survival of liver cancer patients. Taken together, our bioinformatic data-mining analysis provides a comprehensive genomic and transcriptomic landscape of karyopherins in hepatocellular carcinoma and identifies potential members that could be targeted in order to develop new treatment regimens. [ABSTRACT FROM AUTHOR]

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin α in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

In 2011, a hexanucleotide repeat expansion in the first intron of the C9orf72 gene was identified as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The proposed disease mechanisms include loss of C9orf72 function and gain of toxicity from the bidirectionally transcribed repeat-containing RNAs. Over the last few years, substantial progress has been made to determine the contribution of loss and gain of function in disease pathogenesis. The extensive body of molecular, cellular, animal, and human neuropathological studies is conflicted, but the predominance of evidence favors gain of toxicity as the main pathogenic mechanism for C9orf72 repeat expansions. Alterations in several downstream cellular functions, such as nucleocytoplasmic transport and autophagy, are implicated. Exciting progress has also been made in therapy development targeting this mutation, such as by antisense oligonucleotide therapies targeting sense transcripts and small molecules targeting nucleocytoplasmic transport, and these are now in phase 1 clinical trials.

While cytoplasmic aggregation of TDP-43 is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia, how aggregates form and what drives its nuclear clearance have not been determined. Here we show that TDP-43 at its endogenous level undergoes liquid-liquid phase separation (LLPS) within nuclei in multiple cell types. Increased concentration of TDP-43 in the cytoplasm or transient exposure to sonicated amyloid-like fibrils is shown to provoke long-lived liquid droplets of cytosolic TDP-43 whose assembly and maintenance are independent of conventional stress granules. Cytosolic liquid droplets of TDP-43 accumulate phosphorylated TDP-43 and rapidly convert into gels/solids in response to transient, arsenite-mediated stress. Cytoplasmic TDP-43 droplets slowly recruit importin-α and Nup62 and induce mislocalization of RanGap1, Ran, and Nup107, thereby provoking inhibition of nucleocytoplasmic transport, clearance of nuclear TDP-43, and cell death. These findings identify a neuronal cell death mechanism that can be initiated by transient-stress-induced cytosolic de-mixing of TDP-43.

The Nuclear Pore Complex (NPC) represents a critical passage through the nuclear envelope for nuclear import and export that impacts nearly every cellular process at some level. Recent technological advances in the form of Auxin Inducible Degron (AID) strategies and Single-Point Edge-Excitation sub-Diffraction (SPEED) microscopy have enabled us to provide new insight into the distinct functions and roles of nuclear basket nucleoporins (Nups) upon nuclear docking and export for mRNAs. In this paper, we provide a review of our recent findings as well as an assessment of new techniques, updated models, and future perspectives in the studies of mRNA’s nuclear export.