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Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing., (© 2015 Jacinto et al.; Published by Cold Spring Harbor Laboratory Press.)

Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98-Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98-Nup96 joint molecule as a sole protein product of the nup189 (+) gene in the fission yeast Schizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98-Nup96 is dispensable for cell growth, at least under normal culture conditions in S. pombe.

DNA viruses undertake their replication within the cell nucleus, and therefore they must first deliver their genome into the nucleus of their host cells. Thus, trafficking across the nuclear envelope is at the basis of DNA virus infections. Nuclear transport of molecules with diameters up to 39 nm is a tightly regulated process that occurs through the nuclear pore complex (NPC). Due to the enormous diversity of virus size and structure, each virus has developed its own strategy for entering the nucleus of their host cells, with no two strategies alike. For example, baculoviruses target their DNA-containing capsid to the NPC and subsequently enter the nucleus intact, while the hepatitis B virus capsid crosses the NPC but disassembles at the nuclear side of the NPC. For other viruses such as herpes simplex virus and adenovirus, although both dock at the NPC, they have each developed a distinct mechanism for the subsequent delivery of their genome into the nucleus. Remarkably, other DNA viruses, such as parvoviruses and human papillomaviruses, access the nucleus through an NPC-independent mechanism. This review discusses our current understanding of the mechanisms used by DNA viruses to deliver their genome into the nucleus, and further presents the experimental evidence for such mechanisms.

Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate reprogramming.

Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12δ . Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport. [ABSTRACT FROM AUTHOR]

Nucleoporins and the promyelocytic leukemia protein (PML) represent structural entities of nuclear pore complexes and PML nuclear bodies, respectively. In addition, these proteins might function in a common biological mechanism, because at least two different nucleoporins, Nup98 and Nup214, as well as PML, can become aberrantly expressed as oncogenic fusion proteins in acute myeloid leukemia (AML) cells. Here we show that PML and nucleoporins become directed to common cytoplasmic compartments during the mitosis-to-G1 transition of the cell cycle. These protein assemblies, which we have termed CyPNs (cytoplasmic assemblies of PML and nucleoporins), move on the microtubular network and become stably connected to the nuclear membrane once contact with the nucleus has been made. The ability of PML to target CyPNs depends on its nuclear localization signal, and loss of PML causes an increase in cytoplasmic-bound versus nuclear-membrane-bound nucleoporins. CyPNs are also targeted by the acute promyelocytic leukemia (APL) fusion protein PML-RARα and can be readily detected within the APL cell line NB4. These results provide insight into a dynamic pool of cytoplasmic nucleoporins that form a complex with the tumor suppressor protein PML during the G1 phase of the cell cycle. [ABSTRACT FROM AUTHOR]

CRM1-mediated protein export is an important determinant of the nuclear accumulation of many gene regulators. Here, we show that the NFκB transcription factor Dorsal is a substrate of CRM1 and requires the nucleoporin Nup214 for its nuclear translocation upon signaling. Nup214 bound to CRM1 directly and anchored it to the nuclear envelope. In nup214 mutants CRM1 accumulated in the nucleus and NES-protein export was enhanced. Nup214 formed complexes with Nup88 and CRM1 in vivo and Nup214 protected Nup88 from degradation at the nuclear rim. In turn, Nup88 was sufficient for targeting the complex to the nuclear pores. Overexpression experiments indicated that Nup214 alone attracts a fraction of CRM1 to the nuclear envelope but does not interfere with NES-GFP export. By contrast, overexpression of the Nup214-Nup88 complex trapped CRM1 and Dorsal to cytoplasmic foci and inhibited protein export and immune response activation. We hypothesize that variation in levels of the Nup214-Nup88 complex at the pore changes the amount of NPC-bound CRM1 and influences the relative strength and duration of NFκB signaling responses. [ABSTRACT FROM AUTHOR]

Plant nucleocytoplasmic transport beyond the nuclear envelope is important not only for basic cellular functions but also for growth, development, hormonal signaling, and responses to environmental stimuli. Key components of this transport system include nuclear transport receptors and nucleoporins. The functional and physical interactions between receptors and the nuclear pore in the nuclear membrane are indispensable for nucleocytoplasmic transport. Recently, several groups have reported various plant mutants that are deficient in factors involved in nucleocytoplasmic transport. Here, we summarize the current state of knowledge about nucleocytoplasmic transport in plants, and we review the plant-specific regulation and roles of this process in plants.

The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8-47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC.

Nuclear pore complexes are large macromolecular assemblies that facilitate the nucleocytoplasmic exchange of macromolecules. Because of their intricate composition, membrane association, and sheer size, the integration of various, complementary structure determination approaches is a prerequisite for elucidating their structure. We have recently employed such an integrated strategy to analyze the scaffold structure of the cytoplasmic and nuclear rings of the human nuclear pore complex. In this extra view, we highlight two specific aspects of this work: the power of electron microscopy for bridging different resolution regimes and the importance of post-translational modifications for regulating nucleoporin interactions. We review recent technological developments and give a perspective toward future structure determination approaches.

Mutations in the Lotus japonicus nucleoporin genes, NUP85, NUP133, and NENA (SEH1), lead to defects in plant-microbe symbiotic signaling. The homologous proteins in yeast and vertebrates are part of the conserved NUP84/NUP107-160 subcomplex, which is an essential component of the nuclear pore scaffold and has a pivotal role in nuclear pore complex (NPC) assembly. Loss and down-regulation of NUP84/NUP107-160 members has previously been correlated with a variety of growth and molecular defects, however, in L. japonicus only surprisingly specific phenotypes have been reported. We investigated whether Lotus nup85, nup133, and nena mutants exhibit general defects in NPC composition and distribution. Whole mount immunolocalization confirmed a typical nucleoporin-like localization for NUP133, which was unchanged in the nup85-1 mutant. Severe NPC clustering and aberrations in the nuclear envelope have been reported for Saccharomyces cerevisiae nup85 and nup133 mutants. However, upon transmission electron microscopy analysis of L. japonicus nup85, nup133 and nena, we detected only a slight reduction in the average distances between neighboring NPCs in nup133. Using quantitative immunodetection on protein-blots we observed that loss of individual nucleoporins affected the protein levels of other NUP107-160 complex members. Unlike the single mutants, nup85/nup133 double mutants exhibited severe temperature dependent growth and developmental defects, suggesting that the loss of more than one NUP107-160 member affects basal functions of the NPC.

The nuclear pore complex (NPC) is a massive molecular machine embedded in the nuclear envelope and controlling traffic into and out of the cell nucleus. Here, we describe some of the outstanding research questions concerning the NPC, its assembly and functions. We also discuss recent findings that link the NPC and its immediate surroundings to the process of cellular aging. Scaffold and barrier nucleoporins are two major types of protein building blocks that make up the NPC. Surprisingly, these two groups of nucleoporins differ dramatically in their turnover rates. Recent work identifies some of the scaffold nucleoporins as the most extremely long-lived proteins in rat brain. Some of the consequences of these findings and new open questions arising from them are discussed. We also consider the evidence for a perturbed permeability barrier in nuclei from old cells and the alteration of nuclear transport pathways under stress conditions. Finally, we describe the connection between premature aging syndromes and the nuclear lamina, a filamentous protein network which underlies the nuclear envelope., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Recent studies show that nuclear lamins, the type V intermediate filament proteins, are required for proper building of at least some organs. As the major structural components of the nuclear lamina found underneath the inner nuclear membranes, lamins are ubiquitously expressed in all animal cells. How the broadly expressed lamins support the building of specific tissues is not understood. By studying Drosophila testis, we have uncovered a mechanism by which lamin-B functions in the cyst stem cell (CySC) and its differentiated cyst cell, the cell types known to form the niche/microenvironment for the germline stem cells (GSC) and the developing germ line, to ensure testis organogenesis (1). In this extra view, we discuss some remaining questions and the implications of our findings in the understanding of how the ubiquitous nuclear lamina regulates tissue building in a context-dependent manner.

The conformational preference of a peptide with three phenylalanine-glycine (FG) repeats from the intrinsically disordered domain of nucleoporin 159 (nup159) from the yeast nucleopore complex is studied. Conformational states of this FG-peptide in dimethyl sulfoxide (DMSO), a non-native solvent, are first studied. A solvent exchange scheme is designed and performed to understand how the conformational preferences of the peptide are altered as the solvent shifts from DMSO to water. An ensemble of structures of a 19-residue peptide is determined based on (13)Cα, (1)Hα, and (1)HN chemical shifts and with inter-proton distances. An experimental model is then presented where chemical shifts and amide-proton temperature dependence is probed at changing DMSO to water ratios. These co-solvent experiments provide evidence of a conformational change as the fraction of water increases by the stark change in the behavior of amide protons under varied temperature. This investigation provides a NMR based experimental method in the field of intrinsically disordered proteins to realize conformational transitions from a non-native set of structures (in DMSO) to a native set of disordered conformers (in water)., (Copyright © 2013 Wiley Periodicals, Inc.)

From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of "RAN holes" void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus., (© 2013. Published by Elsevier B.V. All rights reserved.)

The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

Nuclear export of mRNPs is mediated by transport factors such as NXF1 that bind mRNPs and mediate their translocation through the central channel of nuclear pores (NPC) using transient interactions with FG-nucleoporins. A number of nuclear factors enhance the efficiency of this process by concentrating mRNPs at the nuclear face of the pores. Although this enhancement has been explored mainly with the yeast TREX-2 complex, recent work has indicated that mammalian cells employ GANP (Germinal-centre Associated Nuclear Protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1-containing mRNPs to NPCs. GANP is constructed from several domains that show local homology to FG-nucleoporins, the yeast mRNA export factor Sac3p and the mammalian MCM3 acetyltransferase. Whereas yeast TREX-2 is located primarily at nuclear pores, some GANP is located in the nuclear interior in addition to that found at the pores. GANP depletion inhibits bulk mRNA export, resulting in retention of mRNPs and NXF1 in punctate foci within the nucleoplasm, consistent with GANP's being an integral component of the mammalian mRNA export machinery. Here, we discuss the model for GANP function presented in our recent paper and its implications for the mechanism of mRNA export in mammalian cells.

Controlled nucleocytoplasmic trafficking is an important feature for fine-tuning signaling pathways in eukaryotic organisms. Nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups) are essential for the exchange of macromolecules across the nuclear envelope. A recent genetic screen in our laboratory identified a partial loss-of-function mutation in Arabidopsis MOS7/Nup88 that causes defects in basal immunity, Resistance (R) protein-mediated defense and systemic acquired resistance. In Drosophila and mammalian cells, exportin-mediated nuclear export of activated Rel/NFκB transcription factors is enhanced in nup88 mutants resulting in immune response failure. Consistent with Nup88 promoting nuclear retention of NFκB, our functional analyses revealed that MOS7/Nup88 is required for appropriate nuclear accumulation of the autoactivated R protein snc1, as well as the key immune regulators EDS1 and NPR1. These results suggest that controlling the nuclear concentrations of specific immune regulators is fundamental for defining defense outputs.

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.

HIV-1 and other lentiviruses have the unusual capability of infecting nondividing cells, but the mechanism by which they cross an intact nuclear membrane is mysterious. Recent work, including a new study (Lee, K.; Ambrose, Z.; Martin, T.D.; Oztop, I.; Mulky, A.; Julias, J.G.; Vandergraaff, N.; Baumann, J.G.; Wang, R.; Yuen, W. et al. Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe2010, 7, 221-233) confirms that the viral capsid plays a key role in HIV-1 nuclear entry in both dividing and nondividing cells. The identification of mutations in the viral capsid that alter the virus's dependence on host cell nucleoporins represents an important advance in this poorly understood stage of the virus life cycle.

The proper communication between organelles is essential for many aspects of eukaryotic life. The coordination of nuclear and cytoplasmic activities in particular is of pivotal importance and depends on transport in and out of the nucleus. The material which translocates through nuclear pores is diverse; it includes numerous proteins, RNAs and large ribonucleoprotein complexes like ribosomal subunits. To ensure the correct nucleocytoplasmic distribution of these components, appropriate mechanisms have to be in place which control traffic across the nuclear envelope. A growing number of studies support the notion that transport through nuclear pore complexes is intimately linked to cell physiology. As such, it has become evident that changes in the cellular environment, either by externally applied stress, aging or disease, alter nuclear traffic. Due to the progress made in the past few years, we are now beginning to understand these processes at the molecular level. Thus, the concept emerges that stress or disease conditions correlate with signaling events which aim at the nuclear transport apparatus. Here, we summarize results from recent publications that provide evidence for the hypothesis that changes in cell physiology modulate nuclear traffic by targeting multiple transport factors. We propose that this traffic control is at least in part mediated by specific signaling events.

In eukaryotic cells, the exchange of molecules between the genetic material within the nucleus and the cytosol occurs through the Nuclear Pore Complex (NPC), which is a large membrane-embedded assembly composed by multiple proteins named nucleoporins arranged around an aqueous channel. The bi-directional passive diffusion and the active transport of factors across the nuclear envelope are responsible for a variety of biological processes and they are controlled respectively by the size of the pore and the interaction between nucleoporins and karyopherins. Thus, it is not surprising that most of the degenerative programs induce cellular stress by altering the NPC composition or the binding between nucleoporins and docking factors. This facilitates the access of nuclear DNA to pro-death factors, amplify the detrimental cascade and finally play a role in the disassembly of the nuclear structure. Recently, we have shown that during calcium-mediated neuronal degeneration NPC components can be degraded with consequent increase of NPC channel permeability. Moreover, we proved that these changes occurred much earlier than the final disassembly of the nuclear envelope and they are mediated by calcium overload. Is the increase of NPC leakiness the executioner of the excitotoxic process or simply a final event of a cell condemned to death? Here we speculate the consequence of the nucleoporin loss, the alteration of nucleocytoplasmic transport and their contribution to neuronal demise.

Separation of DNA replication and transcription, which occur in the nucleus, from protein synthesis, which occurs in the cytoplasm, allows a more precise regulation of these processes. Selective exchange of macromolecules between the two compartments is mediated by proteins of the nuclear pore complex (NPC). Receptor proteins of the karyopherin family interact with NPC components and transfer their cargos between the nucleus and cytoplasm. Nucleocytoplasmic transport pathways are regulated at multiple levels by modulating the expression or function of individual cargoes, transport receptors, or the transport channel. The regulatory levels have increasingly broad effects on the transport pathways and affect a wide range of processes from gene expression to development and differentiation., (© Pleiades Publishing, Ltd. 2010.)