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Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a welldefined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE. [ABSTRACT FROM AUTHOR]

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading, and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT. [ABSTRACT FROM AUTHOR]

In fission yeast lacking the telomere binding protein, Taz1, replication forks stall at telomeres, triggering deleterious downstream events. Strand invasion from one taz1Δ telomeric stalled fork to another on a separate (nonsister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32°C; however, entanglement resolution fails at ≤20°C, leading to cold-specific lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genome-wide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1 Δ and rif1 Δ. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of the nuclear pore complex (NPC) promotes telomere disentanglement and suppresses taz1Δ cold sensitivity. We attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Loss of genes involved in lipid metabolism reverses the ability of rif1 + deletion to suppress taz1Δ cold sensitivity, again pinpointing NE modulation. A rif1 + separation-of-function mutant that specifically loses Rif1's mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1Δ lethality stems from altered NE homeostasis. [ABSTRACT FROM AUTHOR]

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup–Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier. Synopsis: Nuclear pores complexes (NPCs) conduct nucleocytoplasmic transport in eukaryotic cells. This work introduces a nanobody toolkit for studying NPC biogenesis and describes a checkpoint that links assembly of the NPC scaffold to that of the NPC permeability barrier. Direct tracking of postmitotic NPC assembly with fluorescent nanobodies that recognize well-accessible Nup epitopes. Novel interphase NPC assembly assay using frog-specific anti-Nup nanobodies to track the insertion of new frog NPCs into a human template nucleus. Nanobodies that block Nup-Nup interactions and inhibit NPC assembly. Structures of Nup complexes with inhibitory nanobodies. Inhibitory anti-Nup98 nanobodies identify a checkpoint that avoids non-selective NPCs without a permeability barrier. A nanobody toolkit for tracking or blocking nuclear pore complex (NPC) biogenesis reveals that assembly of the NPC scaffold is linked to formation of a functional permeability barrier. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) has multiple functions beyond the nucleo-cytoplasmic transport of large molecules. Subnuclear compartmentalization of chromatin is critical for gene expression in animals and yeast. However, the mechanism by which the NPC regulates gene expression is poorly understood in plants. Here we report that the Y-complex (Nup107–160 complex, a subcomplex of the NPC) self-maintains its nucleoporin homeostasis and modulates FLOWERING LOCUS C (FLC) transcription via changing histone modifications at this locus. We show that Y-complex nucleoporins are intimately associated with FLC chromatin through their interactions with histone H2A at the nuclear membrane. Fluorescence in situ hybridization assays revealed that Nup96, a Y-complex nucleoporin, enhances FLC positioning at the nuclear periphery. Nup96 interacted with HISTONE DEACETYLASE 6 (HDA6), a key repressor of FLC expression via histone modification, at the nuclear membrane to attenuate HDA6-catalyzed deposition at the FLC locus and change histone modifications. Moreover, we demonstrate that Y-complex nucleoporins interact with RNA polymerase II to increase its occupancy at the FLC locus, facilitating transcription. Collectively, our findings identify an attractive mechanism for the Y-complex in regulating FLC expression via tethering the locus at the nuclear periphery and altering its histone modification. The nuclear pore Y-complex acts as a functional compartmentalization platform for histone epigenetic modification at the FLOWERING LOCUS C locus conferring flowering regulation in Arabidopsis. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain. [ABSTRACT FROM AUTHOR]

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIV's uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also confirmed that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associated with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like the complex retrovirus HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells. Author summary: Retrovirus replication requires nuclear entry of reverse-transcribed double-stranded viral DNA and integration into the host cell chromosomes. It is widely believed that unlike lentiviruses, simple retroviruses like MLV require nuclear membrane breakdown during cell division to achieve nuclear entry of viral reverse transcription products to complete their replication. We show here that while this is true for rapidly dividing tissue culture cells, in quiescent primary cells like dendritic cells, the likely initial targets of in vivo MLV infection, the virus establishes infection without cell division. MLV accomplishes this because the viral capsid protein interacts with proteins in the nuclear membrane in DCs. In contrast, while this interaction occurs in dividing tissue culture cells, it is not sufficient for nuclear entry of viral DNA. These studies show that the requirements for retrovirus infection depend on the cell type and moreover, partially overturn the dogma that simple retroviruses need cell division to complete replication. [ABSTRACT FROM AUTHOR]

Nucleoporin 50 (Nup50) is an evolutionarily conserved protein that is a constituent of the nuclear pore complex (NPC); however, its physiological role in plants is unclear. Arabidopsis has two Nup50 proteins, Nup50a and Nup50b, which are highly expressed in developing seeds. Green fluoresceent protein (GFP)-fused Nup50a and Nup50b are localized exclusively in the nucleopolasm, implying an additional function beyond the NPC in the nuclear envelope. To investigate the function of Nup50s, we employed the CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] system to generate a nup50a nup50b double mutant, which exhibited premature translation termination of both Nup50 proteins. While the mutant showed no significant abnormal phenotype during vegetative growth, the nup50a nup50b seeds had an abnormal shape compared with the wild type. Comparative transcriptomics using immature seeds revealed that Nup50s regulate the expression of various genes, including cell wall-related genes. The nup50a nup50b seeds exhibited reduced seed longevity and salinity stress tolerance. Tetrazolium uptake and mucilage release assays implied that the nup50a nup50b seeds had greater water permeability than the wild type. Taken together, our results imply that Nup50s play a critical role in seed formation by regulating gene expression. [ABSTRACT FROM AUTHOR]

Nuclear Pore Complexes (NPCs) are embedded in the nuclear envelope (NE), regulating macromolecule transport and physically interacting with chromatin. The NE undergoes dramatic breakdown and reformation during plant cell division. In addition, this structure has a specific meiotic function, anchoring and positioning telomeres to facilitate the pairing of homologous chromosomes. To elucidate a possible function of the structural components of the NPCs in meiosis, we have characterized several Arabidopsis lines with mutations in genes encoding nucleoporins belonging to the outer ring complex. Plants defective for either SUPPRESSOR OF AUXIN RESISTANCE1 (SAR1, also called NUP160) or SAR3 (NUP96) present condensation abnormalities and SPO11-dependent chromosome fragmentation in a fraction of meiocytes, which is increased in the double mutant sar1 sar3. We also observed these meiotic defects in mutants deficient in the outer ring complex protein HOS1, but not in mutants affected in other components of this complex. Furthermore, our findings may suggest defects in the structure of NPCs in sar1 and a potential link between the meiotic role of this nucleoporin and a component of the RUBylation pathway. These results provide the first insights in plants into the role of nucleoporins in meiotic chromosome behavior. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) has long been assumed to be the sole route across the nuclear envelope, and under normal homeostatic conditions it is indeed the main mechanism of nucleo-cytoplasmic transport. However, it has also been known that e.g. herpesviruses cross the nuclear envelope utilizing a pathway entitled nuclear egress or envelopment/de-envelopment. Despite this, a thread of observations suggests that mechanisms similar to viral egress may be transiently used also in healthy cells. It has since been proposed that mechanisms like nuclear envelope budding (NEB) can facilitate the transport of RNA granules, aggregated proteins, inner nuclear membrane proteins, and mis-assembled NPCs. Herein, we will summarize the known roles of NEB as a physiological and intrinsic cellular feature and highlight the many unanswered questions surrounding these intriguing nuclear events. [ABSTRACT FROM AUTHOR]

Eukaryotic cells organize their genome within the nucleus with a double-layered membrane structure termed the nuclear envelope (NE) as the physical barrier. The NE not only shields the nuclear genome but also spatially separates transcription from translation. Proteins of the NE including nucleoskeleton proteins, inner nuclear membrane proteins, and nuclear pore complexes have been implicated in interacting with underlying genome and chromatin regulators to establish a higher-order chromatin architecture. Here, I summarize recent advances in the knowledge of NE proteins that are involved in chromatin organization, gene regulation, and coordination of transcription and mRNA export. These studies support an emerging view of plant NE as a central hub that contributes to chromatin organization and gene expression in response to various cellular and environmental cues. [ABSTRACT FROM AUTHOR]

Introduction: Plasmid DNA (pDNA) must be delivered into the nucleus for transgene expression in mammalian cells. The entry may happen passively during the nuclear envelope breakdown and reformation in dividing cells or actively through the nuclear pore complexes. The goal of this study was to investigate the relative importance of these two pathways for pDNA nuclear entry and subsequent gene expression. Methods: To measure nuclear entry of pDNA encoding enhanced green florescence protein (EGFP) in electrotransfected cells, we developed a sensitive technique for quantitative analysis of pDNA in the nuclei, based on a hybridization probe for pDNA detection at the single molecule level and automatic image analysis. In matched experiments, we used an mRNA targeted hybridization probe to quantify reporter mRNA expression per cell, and flow cytometry to quantify expression of EGFP. Results: We discovered two distinct patterns of pDNA distribution in the nuclei: punctate and diffuse, which were dominant in arrested and unarrested cells, respectively. The cell cycle arrest decreased diffuse pDNA and increased punctate pDNA. Its net effect was a decrease in the total intranuclear pDNA. Additionally, the cell cycle arrest increased the reporter mRNA synthesis but had no substantial impact on reporter protein expression. Conclusion: Results from the study demonstrated that the efficient nuclear entry of pDNA during cell division did not necessarily lead to a high level of transgene expression. They also suggested that the punctate pDNA was more transcriptionally active than diffuse pDNA in the nuclei. These data will be useful in future studies for understanding mechanisms of nonviral gene delivery. [ABSTRACT FROM AUTHOR]

The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear shape and gene expression. To understand how the Arabidopsis (Arabidopsis thaliana) nucleoskeleton physically connects to the nuclear periphery in plants, we investigated the Arabidopsis nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. We identified 3 conserved peptide motifs within the N-terminal region of KAKU4, which are required for intermolecular interactions of KAKU4 with itself, interaction with the nucleoskeleton protein CROWDED NUCLEI (CRWN), localization at the nuclear periphery, and nuclear elongation in differentiated tissues. Unexpectedly, we find these motifs to be present also in NUP82 and NUP136, 2 plant-specific nucleoporins from the nuclear pore basket. We further show that NUP82, NUP136, and KAKU4 have a common evolutionary history predating nonvascular land plants with KAKU4 mainly localizing outside the nuclear pore suggesting its divergence from an ancient nucleoporin into a new nucleoskeleton component. Finally, we demonstrate that both NUP82 and NUP136, through their shared N-terminal motifs, interact with CRWN and KAKU4 proteins revealing the existence of a physical continuum between the nuclear pore and the nucleoskeleton in plants. The KAKU4 protein, a component of the plant nucleoskeleton, diverged from an ancestral nucleoporin and provides a continuum between the nucleoskeleton and the nuclear pore complex basket via short peptide motifs. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-β plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis. [ABSTRACT FROM AUTHOR]

The karyosphere (karyosome) is a structure that forms in the oocyte nucleus—germinal vesicle (GV)—at the diplotene stage of meiotic prophase due to the assembly of all chromosomes in a limited portion of the GV. In some organisms, the karyosphere has an extrachromosomal external capsule, the marker protein of which is nuclear F-actin. Despite many years of theories about the formation of the karyosphere capsule (KC) in the GV of the common frog Rana temporaria, we present data that cast doubt on its existence, at least in this species. Specific extrachromosomal strands, which had been considered the main elements of the frog's KC, do not form a continuous layer around the karyosphere and, according to immunogold labeling, do not contain structural proteins, such as actin and lamin B. At the same time, F-actin is indeed noticeably concentrated around the karyosphere, creating the illusion of a capsule at the light microscopy/fluorescence level. The barrier-to-autointegration factor (BAF) and one of its functional partners—LEMD2, an inner nuclear membrane protein—are not localized in the strands, suggesting that the strands are not functional counterparts of the nuclear envelope. The presence of characteristic strands in the GV of R. temporaria late oocytes may reflect an excess of SMC1 involved in the structural maintenance of diplotene oocyte chromosomes at the karyosphere stage, since SMC1 has been shown to be the most abundant protein in the strands. Other characteristic microstructures—the so-called annuli, very similar in ultrastructure to the nuclear pore complexes—do not contain nucleoporins Nup35 and Nup93, and, therefore, they cannot be considered autonomous pore complexes, as previously thought. Taken together, our data indicate that traditional ideas about the existence of the R. temporaria KC as a special structural compartment of the GV are to be revisited. [ABSTRACT FROM AUTHOR]

The integrity of the nuclear envelope depends on the function of nuclear pore complexes (NPCs), transport channels that control macromolecular traffic between the nucleus and cytosol. The central importance of NPCs suggests the existence of quality control (QC) mechanisms that oversee their assembly and function. In this perspective, we emphasize the challenges associated with NPC assembly and the need for QC mechanisms that operate at various stages of an NPC's life. This includes cytosolic preassembly QC that helps enforce key nucleoporin–nucleoporin interactions and their ultimate stoichiometry in the NPC in addition to mechanisms that monitor aberrant fusion of the inner and outer nuclear membranes. Furthermore, we discuss whether and how these QC mechanisms may operate to sense faulty mature NPCs to facilitate their repair or removal. The so far uncovered mechanisms for NPC QC provide fertile ground for future research that not only benefits a better understanding of the vital role that NPCs play in cellular physiology but also how loss of NPC function and/or these QC mechanisms might be an input to aging and disease. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes are large multicomponent protein complexes that are embedded in the nuclear envelope, where they mediate nucleocytoplasmic transport. In addition to supporting transport, nuclear pore components, termed nucleoporins (Nups), can interact with chromatin and influence genome function. A subset of Nups can also localize to the nuclear interior and bind chromatin intranuclearly, providing an opportunity to investigate chromatin‐associated functions of Nups outside of the transport context. This review focuses on the gene regulatory functions of such intranuclear Nups, with a particular emphasis on their identity as components of several chromatin regulatory complexes. Recent proteomic screens have identified Nups as interacting partners of active and repressive epigenetic machinery, architectural proteins, and DNA replication complexes, providing insight into molecular mechanisms via which Nups regulate gene expression programs. This review summarizes these interactions and discusses their potential functions in the broader framework of nuclear genome organization. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are sophisticated multiprotein assemblies embedded within the nuclear envelope and controlling the exchanges of molecules between the cytoplasm and the nucleus. In this review, we summarize the mechanisms by which these elaborate complexes are built from their subunits, the nucleoporins, based on our ever‐growing knowledge of NPC structural organization and on the recent identification of additional features of this process. We present the constraints faced during the production of nucleoporins, their gathering into oligomeric complexes, and the formation of NPCs within nuclear envelopes, and review the cellular strategies at play, from co‐translational assembly to the enrolment of a panel of cofactors. Remarkably, the study of NPCs can inform our perception of the biogenesis of multiprotein complexes in general – and vice versa. [ABSTRACT FROM AUTHOR]

Heterochromatin and euchromatin form different spatial compartments in the interphase nucleus, with heterochromatin being localized mainly at the nuclear periphery. The mechanisms responsible for peripheral localization of heterochromatin are still not fully understood. The nuclear lamina and nuclear pore complexes were obvious candidates for the role of heterochromatin binders. This review is focused on recent studies showing that heterochromatin interactions with the nuclear lamina and nuclear pore complexes maintain its peripheral localization. Differences in chromatin interactions with the nuclear envelope in cell populations and in individual cells are also discussed. [ABSTRACT FROM AUTHOR]

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

The Amoebozoan Dictyostelium discoideum exhibits a semi-closed mitosis in which the nuclear membranes remain intact but become permeabilized to allow tubulin and spindle assembly factors to access the nuclear interior. Previous work indicated that this is accomplished at least by partial disassembly of nuclear pore complexes (NPCs). Further contributions by the insertion process of the duplicating, formerly cytosolic, centrosome into the nuclear envelope and nuclear envelope fenestrations forming around the central spindle during karyokinesis were discussed. We studied the behavior of several Dictyostelium nuclear envelope, centrosomal, and nuclear pore complex (NPC) components tagged with fluorescence markers together with a nuclear permeabilization marker (NLS-TdTomato) by live-cell imaging. We could show that permeabilization of the nuclear envelope during mitosis occurs in synchrony with centrosome insertion into the nuclear envelope and partial disassembly of nuclear pore complexes. Furthermore, centrosome duplication takes place after its insertion into the nuclear envelope and after initiation of permeabilization. Restoration of nuclear envelope integrity usually occurs long after re-assembly of NPCs and cytokinesis has taken place and is accompanied by a concentration of endosomal sorting complex required for transport (ESCRT) components at both sites of nuclear envelope fenestration (centrosome and central spindle). [ABSTRACT FROM AUTHOR]

The nucleus is characteristic of eukaryotic cells and nuclear envelope proteins are conserved across the kingdoms. Over the years, the function of these proteins was studied in the intact nuclear envelope. Knowledge regarding the localization and function of nuclear envelope proteins during mitosis, after the nuclear envelope breaks down, is limited. Until recently, the localization of nuclear envelope proteins during mitosis has been observed with the mitotic apparatus. In this context, research in plant cell biology is more advanced compared to non-plant model systems. Although current studies shed light on the localization of nuclear envelope proteins, further experiments are required to determine what, if any, functional role different nuclear envelope proteins play during mitosis. This review will highlight our current knowledge about the role of nuclear envelope proteins and point out the unanswered questions as future direction. [ABSTRACT FROM AUTHOR]

The nuclear envelope (NE) protects but also organizes the eukaryotic genome. In this review we will discuss recent literature on how the NE disassembles and reassembles, how it varies in surface area and protein composition and how this translates into chromatin organization and gene expression in the context of animal development. [ABSTRACT FROM AUTHOR]

A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localization studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution. [ABSTRACT FROM AUTHOR]

A recent article in The Plant Cell explores the role of nuclear pore complexes in regulating gene expression in plant cells. While previous research has shown that nuclear pore complexes can affect gene expression in mammalian cells, their impact on plant cells has been understudied. The study found that a specific nuclear pore subcomplex, known as the Y-complex, physically moves the FLOWERING LOCUS C (FLC) locus closer to the periphery of the nucleus in Arabidopsis, resulting in upregulated gene expression. The authors also discovered that members of the Y-complex interact with histones and RNA Polymerase II, providing insights into the mechanism behind this regulation. The findings raise questions about the factors that regulate FLC-Y-complex tethering, such as vernalization, photoperiod, circadian clock control, and stress resilience. [Extracted from the article]

The nuclear envelope is a unique subdomain of the endoplasmic reticulum (ER) that encapsulates the genome and mediates communication between the nucleus and the rest of the cell via nuclear pore complexes. A recent study by Romanauska and Köhler shows that balanced lipid unsaturation is critical for nuclear envelope and nuclear pore complex architecture and function. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle. [ABSTRACT FROM AUTHOR]

In eukaryotes, the nucleus is the regulatory center of cytogenetics and metabolism, and it is critical for fundamental biological processes, including DNA replication and transcription, protein synthesis, and biological macromolecule transportation. The eukaryotic nucleus is surrounded by a lipid bilayer called the nuclear envelope (NE), which creates a microenvironment for sophisticated cellular processes. The NE is perforated by the nuclear pore complex (NPC), which is the channel for biological macromolecule bi-directional transport between the nucleus and cytoplasm. It is well known that NPC is the spatial designer of the genome and the manager of genomic function. Moreover, the NPC is considered to be a platform for the continual adaptation and evolution of eukaryotes. So far, a number of nucleoporins required for plant-defense processes have been identified. Here, we first provide an overview of NPC organization in plants, and then discuss recent findings in the plant NPC to elaborate on and dissect the distinct defensive functions of different NPC subcomponents in plant immune defense, growth and development, hormone signaling, and temperature response. Nucleoporins located in different components of NPC have their unique functions, and the link between the NPC and nucleocytoplasmic trafficking promotes crosstalk of different defense signals in plants. It is necessary to explore appropriate components of the NPC as potential targets for the breeding of high-quality and broad spectrum resistance crop varieties. [ABSTRACT FROM AUTHOR]

Nuclear pore complex (NPC) embedded in the nuclear envelope, is the only channel for macromolecule nucleocytoplasmic transportation and has important biological functions. However, the deregulation of specific nucleoporins (Nups) and NPC-Nup-based mechanisms and their function in tumour progression remain poorly understood. Here, we aimed to identify the Nups that contribute to HCC progression and metastasis in 729 primary hepatocellular carcinoma (HCC) cases using molecular, cytological, and biochemical techniques. Our results revealed elevated Nup93 expression in HCC tissues, especially in cases with metastasis, and was linked to worse prognosis. Furthermore, Nup93 knockdown suppressed HCC cell metastasis and proliferation, while Nup93 overexpression promoted these activities. We observed that Nup93 promotes HCC metastasis and proliferation by regulating β-catenin translocation. In addition, we found that Nup93 interacted with β-catenin directly, independent of importin. Furthermore, LEF1 and β-catenin facilitated the Nup93-mediated metastasis and proliferation in HCC via a positive feedback loop. Thus, our findings provide novel insights into the mechanisms underlying the Nup93-induced promotion of HCC metastasis and suggest potential therapeutic targets in the LEF1-Nup93-β-catenin pathway for HCC therapeutics. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are huge protein assemblies within the nuclear envelope (NE) that serve as selective gates for macromolecular transport between nucleus and cytoplasm. When higher eukaryotic cells prepare for division, they rapidly disintegrate NPCs during NE breakdown such that nuclear and cytoplasmic components mix to enable the formation of a cytoplasmic mitotic spindle. At the end of mitosis, reassembly of NPCs is coordinated with the establishment of the NE around decondensing chromatin. We review recent progress on mitotic NPC disassembly and reassembly, focusing on vertebrate cells. We highlight novel mechanistic insights into how NPCs are rapidly disintegrated into conveniently reusable building blocks, and put divergent models of (post-)mitotic NPC assembly into a spatial and temporal context. NPC disintegration requires the combined activity of multiple mitotic kinases and exploits multisite phosphorylation, linking it to mitotic commitment. NPC disassembly generates smaller building blocks that are conveniently reusable during mitotic exit, and some of these may even remain membrane-associated throughout mitosis and serve as templates for NPC reassembly. Enclosure of all chromatin into a single nucleus that contains transport-competent NPCs requires chromosome clustering and the coordination of NPC assembly and NE reformation. Mitotic NPC reassembly starts at lateral areas of the decondensing chromatin mass, away from spindle microtubules, possibly by attraction of perforated endoplasmic reticulum (ER) membrane sheets to defined NPC assembly sites on chromatin. NPC reassembly during mitotic exit may encompass a range of modes whose prevalence is spatially and temporarily determined. [ABSTRACT FROM AUTHOR]

Membrane-bound organelles provide physical and functional compartmentalization of biological processes in eukaryotic cells. The characteristic shape and internal organization of these organelles is determined by a combination of multiple internal and external factors. The maintenance of the shape of nucleus, which houses the genetic material within a double membrane bilayer, is crucial for a seamless spatio-temporal control over nuclear and cellular functions. Dynamic morphological changes in the shape of nucleus facilitate various biological processes. Chromatin packaging, nuclear and cytosolic protein organization, and nuclear membrane lipid homeostasis are critical determinants of overall nuclear morphology. As such, a multitude of molecular players and pathways act together to regulate the nuclear shape. Here, we review the known mechanisms governing nuclear shape in various unicellular and multicellular organisms, including the non-spherical nuclei and non-lamin-related structural determinants. The review also touches upon cellular consequences of aberrant nuclear morphologies. [ABSTRACT FROM AUTHOR]

The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions. Chromatin mobility contributes to the homology directed repair (HDR) of double strand breaks (DSBs) and replication stress, with some DNA lesions specifically translocated to nuclear hubs of HDR activity. Polymerization of nucleus-specific filamentous action (F-actin) promotes DSB and stalled replication fork mobility, including the directed movement of lesions along F-actin to the nuclear periphery. Nuclear pore complexes (NPC) participate in the HDR of stalled replication forks, eroded telomeres, and persistent DSBs, potentially through the break‐induced‐replication (BIR) pathway. In the 10% of human tumors that utilize alternative lengthening of telomeres (ALT), chromosome ends are mobilized to promyelocytic leukemia protein nuclear bodies (PML-NBs), which function as hubs of telomere HDR activity. [ABSTRACT FROM AUTHOR]

The hallmark of eukaryotic cells is the nucleus that contains the genome, enclosed by a physical barrier known as the nuclear envelope (NE). On the one hand, this compartmentalization endows the eukaryotic cells with high regulatory complexity and flexibility. On the other hand, it poses a tremendous logistic and energetic problem of transporting millions of molecules per second across the nuclear envelope, to facilitate their biological function in all compartments of the cell. Therefore, eukaryotes have evolved a molecular "nanomachine" known as the Nuclear Pore Complex (NPC). Embedded in the nuclear envelope, NPCs control and regulate all the bi-directional transport between the cell nucleus and the cytoplasm. NPCs combine high molecular specificity of transport with high throughput and speed, and are highly robust with respect to molecular noise and structural perturbations. Remarkably, the functional mechanisms of NPC transport are highly conserved among eukaryotes, from yeast to humans, despite significant differences in the molecular components among various species. The NPC is the largest macromolecular complex in the cell. Yet, despite its significant complexity, it has become clear that its principles of operation can be largely understood based on fundamental physical concepts, as have emerged from a combination of experimental methods of molecular cell biology, biophysics, nanoscience and theoretical and computational modeling. Indeed, many aspects of NPC function can be recapitulated in artificial mimics with a drastically reduced complexity compared to biological pores. We review the current physical understanding of the NPC architecture and function, with the focus on the critical analysis of experimental studies in cells and artificial NPC mimics through the lens of theoretical and computational models. We also discuss the connections between the emerging concepts of NPC operation and other areas of biophysics and bionanotechnology. [ABSTRACT FROM AUTHOR]

Nuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described. [ABSTRACT FROM AUTHOR]

The defining feature of the eukaryotic cell, the nucleus, is bounded by a double envelope. This envelope and the nuclear pores within it play a critical role in separating the genome from the cytoplasm. It also presents cells with a challenge. How are cells to remodel the nuclear compartment boundary during mitosis without compromising nuclear function? In the two billion years since the emergence of the first cells with a nucleus, eukaryotes have evolved a range of strategies to do this. At one extreme, the nucleus is disassembled upon entry into mitosis and then reassembled anew in the two daughter cells. At the other, cells maintain an intact nuclear compartment boundary throughout the division process. In this review, we discuss common features of the division process that underpin remodelling mechanisms, the topological challenges involved and speculate on the selective pressures that may drive the evolution of distinct modes of division. [ABSTRACT FROM AUTHOR]

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

Nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus. This NLS-dependent protein recognition, a process necessary for cargo proteins to pass the nuclear envelope through the nuclear pore complex, is facilitated by members of the importin superfamily. Here, we summarized the types of NLS, focused on the recently reported related proteins containing nuclear localization signals, and briefly summarized some mechanisms that do not depend on nuclear localization signals into the nucleus. 9b35b5XrF5YBVYeQB6JADF Video Abstract [ABSTRACT FROM AUTHOR]