Nuclear Pore Complex Function Research Articles

Nucleoporin93 limits Yap activity to prevent endothelial cell senescence.

As the innermost lining of the vasculature, endothelial cells (ECs) are constantly subjected to systemic inflammation and particularly vulnerable to aging. Endothelial health is hence vital to prevent age-related vascular disease. Healthy ECs rely on the proper localization of transcription factors via nuclear pore complexes (NPCs) to govern cellular behavior. Emerging studies report NPC degradation with natural aging, suggesting impaired nucleocytoplasmic transport in age-associated EC dysfunction. We herein identify nucleoporin93 (Nup93), a crucial structural NPC protein, as an indispensable player in vascular protection. Endothelial Nup93 protein levels are significantly reduced in the vasculature of aged mice, paralleling observations of Nup93 loss when using invitro models of EC senescence. The loss of Nup93 in human ECs induces cell senescence and promotes the expression of inflammatory adhesion molecules, where restoring Nup93 protein in senescent ECs reverses features of endothelial aging. Mechanistically, we find that both senescence and loss of Nup93 impair endothelial NPC transport, leading to nuclear accumulation of Yap and downstream inflammation. Pharmacological studies indicate Yap hyperactivation as the primary consequence of senescence and Nup93 loss in ECs. Collectively, our findings indicate that the maintenance of endothelial Nup93 is a key determinant of EC health, where aging targets endothelial Nup93 levels to impair NPC function as a novel mechanism of EC senescence and vascular aging.

Changing the guard-nuclear pore complex quality control.

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.

TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei.

ABSTRACTTwenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups — Nup176, 269, 335, 390, and 434 — are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position.

441 Super-resolution imaging for nuclear pore quantification in human keratinocyte differentiation

Nuclear Pore Complexes (NPCs) serve as essential gateways between the nucleus and cytoplasm. Dysregulation of NPC function is associated with aging and cancer, yet how NPC is implicated in epidermal homeostasis and skin disorders remains largely unexplored. Here, we show that the 74% of genes encoding nucleoporins (NUPs), the building blocks constituting the NPC, are significantly downregulated (p<0.05) in primary human keratinocyte differentiation. NUP93, an essential NUP for determining the protein diffusion size limit across the nuclear pores, is among the most drastically downregulated NUPs.

Architecture of the cytoplasmic face of the nuclear pore.

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y‑shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment‑specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease‑associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell‑based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled‑coil hub that tethers two separate mRNP‑remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan‑specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N‑terminal S‑shaped α‑helical solenoid followed by a coiled‑coil oligomerization element, numerous Ran‑interacting domains, an E3 ligase domain, and a C‑terminal prolyl‑isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N‑terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell‑based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo‑ET density matched the dimensions of the CFNC coiled‑coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled‑coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo‑ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near‑atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].

Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].

AI-based structure prediction empowers integrative structural analysis of human nuclear pores.

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

A Nucleoporin NUP58 modulates responses to drought and salt stress in maize (Zea mays L.)

Nuclear pore complex (NUP) is the main transport channel between cytoplasm and nucleoplasm, which plays an important role in stress response. The function of NUPs was widely reported in yeast and vertebrate but rarely in plants. Here, we identified a nuclear pore complex (ZmNUP58), that is tightly associated with drought and salt tolerance phenotype accompanied with phenotypic and physiological changes under drought and salt stress. The overexpression of ZmNUP58 in maize (Zea mays L.) significantly promotes both chlorophyll content and activities of antioxidant enzymes under drought- and salt-stressed conditions. RNA-Seq analysis showed that ZmNUP58 could regulate the expression of genes related to phytohormone synthesis and signaling, osmotic adjustment substances, antioxidant enzyme system, cell wall biosynthesis, glucose metabolism and aquaporin. The results provide novel insights into the regulatory role of ZmNUP58 in improving drought and salt tolerance through regulating phytohormone and other stress response genes in maize.

Function of Nuclear Pore Complexes in Regulation of Plant Defense Signaling.

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.

Uip4p modulates nuclear pore complex function in Saccharomyces cerevisiae

ABSTRACT 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.

Analysis of Nuclear Pore Complex Permeability in Mammalian Cells and Isolated Nuclei Using Fluorescent Dextrans.

In eukaryotic cells the nuclear envelope encloses the genome separating it from the rest of the cell. Nuclear pore complexes are large multi protein channels that perforate the nuclear envelope, connecting the nucleus and the cytoplasm. Besides controlling nucleocytoplasmic molecule exchange, nuclear pore complexes create a permeability barrier that defines the maximum size of molecules that can freely diffuse into the nucleus. Accumulating evidence indicate that the permeability barrier of the nucleus can vary in different cellular conditions, during aging and in disease. Here we provide a simple protocol to analyze changes in nuclear permeability in plasma membrane-permeabilized cells and isolated nuclei using fluorescent dextrans of different sizes and confocal microscopy. The methods described herein represent a valuable resource to researchers studying the function of nuclear pore complexes and the dynamics of nuclear permeability in different cell types and processes.

Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology During Confined Migration.

Cancer metastasis, that is, the spreading of tumor cells from the primary tumor to distant sites, requires cancer cells to travel through pores substantially smaller than their cross section . This "confined migration" requires substantial deformation by the relatively large and rigid nucleus, which can impact nuclear compartmentalization, trigger cellular mechanotransduction pathways, and increase genomic instability. To improve our understanding of how cells perform and respond to confined migration, we developed polydimethylsiloxane (PDMS) microfluidic devices in which cells migrate through a precisely controlled "field of pillars" that closely mimic the intermittent confinement of tumor microenvironments and interstitial spaces. The devices can be designed with various densities of pillars, ranging from a very low density that does not require nuclear deformation to high densities that present microenvironment conditions with severe confinement. The devices enable assessment of cellular fitness for confined migration based on the distance traveled through the constriction area over several days. In this protocol, we present two complementary techniques to generate silicon master molds for the device fabrication: (1) SU-8 soft lithography for rapid prototyping and for devices with relatively large features; and (2) reactive ion etching (RIE) to achieve finer features and more durable molds. In addition, we describe the production, use, and validation of the devices, along with the analysis pipeline for experiments using the devices with fluorescently labeled cells. Collectively, this protocol enables the study of confined migration and is readily amendable to investigate other aspects of confined migration mechanobiology, such as nuclear pore complex function in response to nuclear deformation.

Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts.

The nuclear pore complex (NPC) is the conduit in the nuclear envelope through which proteins and RNA are transported between the cytoplasm and nucleus. Xenopus egg extracts that support de novo assembly of nuclei have provided a robust system to study NPC structure and function because the biochemical composition of the extract can be easily manipulated. Here we describe how to assemble nuclei in Xenopus egg extract, how to visualize and analyze NPCs in both live and fixed samples, and different approaches to altering nucleocytoplasmic transport in extract.

Dunking into the Lipid Bilayer: How Direct Membrane Binding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and Assembly.

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

Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS.

Alterations in the components [nucleoporins (Nups)] and function of the nuclear pore complex (NPC) have been implicated as contributors to the pathogenesis of genetic forms of neurodegeneration including C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We hypothesized that Nup alterations and the consequential loss of NPC function may lie upstream of TDP-43 dysfunction and mislocalization widely observed in ALS, FTD, and related neurodegenerative diseases. Here, we provide evidence that CHMP7, a critical mediator of NPC quality control, is increased in nuclei of C9orf72 and sporadic ALS induced pluripotent stem cell (iPSC)-derived spinal neurons (iPSNs) and postmortem human motor cortex before the emergence of Nup alterations. Inhibiting the nuclear export of CHMP7 triggered Nup reduction and TDP-43 dysfunction and pathology in human neurons. Knockdown of CHMP7 alleviated disease-associated Nup alterations, deficits in Ran GTPase localization, defects in TDP-43-associated mRNA expression, and downstream glutamate-induced neuronal death. Thus, our data support a role for altered CHMP7-mediated Nup homeostasis as a prominent initiating pathological mechanism for familial and sporadic ALS and highlight the potential for CHMP7 as therapeutic target.

On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions.

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the β-karyopherin (Kapβ) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapβs, cargoes and Ran to establish the asymmetry of NCT.

Acute Heat Stress Leads to Reversible Aggregation of Nuclear Proteins into Nucleolar Rings in Fission Yeast

Upon acute heat stress (HS), overall mRNA transcription, processing, and export are inhibited, leading to cell growth arrest. However, how cells turn off mRNA metabolism is not fully understood. Here, we show that acute HS results in the segregation and aggregation of multiple nuclear and nucleolar proteins into ring-like structures located at the nucleolar periphery (nucleolar rings [NuRs]). NuRs sequester essential factors required for nuclear mRNA metabolism and nuclear pore complex function, as well as cell-cycle regulators. When cells are switched back to growing temperatures, NuRs disaggregate, and their components relocate to their functional environments in an Hsf1- and Hsp104-dependent manner, and concomitantly with the reinitiation of cell growth. These findings highlight the contribution of reversible protein aggregation to the inhibition of overall RNA-related activities in the nucleus and its functional relevance in the maintenance of cellular homeostasis during acute HS.

The Structure of the Nuclear Pore Complex (An Update).

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

Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer’s Disease

Tau is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD), but the mechanism underlying tau-associated neural damage remains unclear. Here, we show that tau can directly interact with nucleoporins of the nuclear pore complex (NPC) and affect their structural and functional integrity. Pathological tau impairs nuclear import and export in tau-overexpressing transgenic mice and in human AD brain tissue. Furthermore, the nucleoporin Nup98 accumulates in the cell bodies of some tangle-bearing neurons and can facilitate tau aggregation invitro. These data support the hypothesis that tau can directly interact with NPC components, leading to their mislocalization and consequent disruption of NPC function. This raises the possibility that NPC dysfunction contributes to tau-induced neurotoxicity in AD and tauopathies.

TorsinA dysfunction causes persistent neuronal nuclear pore defects.

A critical challenge to deciphering the pathophysiology of neurodevelopmental disease is identifying which of the myriad abnormalities that emerge during CNS maturation persist to contribute to long-term brain dysfunction. Childhood-onset dystonia caused by a loss-of-function mutation in the AAA+ protein torsinA exemplifies this challenge. Neurons lacking torsinA develop transient nuclear envelope (NE) malformations during CNS maturation, but no NE defects are described in mature torsinA null neurons. We find that during postnatal CNS maturation torsinA null neurons develop mislocalized and dysfunctional nuclear pore complexes (NPC) that lack NUP358, normally added late in NPC biogenesis. SUN1, a torsinA-related molecule implicated in interphase NPC biogenesis, also exhibits localization abnormalities. Whereas SUN1 and associated nuclear membrane abnormalities resolve in juvenile mice, NPC defects persist into adulthood. These findings support a role for torsinA function in NPC biogenesis during neuronal maturation and implicate altered NPC function in dystonia pathophysiology.