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Nuclear pore complexes (NPC) are the central mediators of nucleocytoplasmic transport. Increasing evidence shows that many cancer cells have increased numbers of NPCs and become addicted to the nuclear transport machinery. How reducing NPC numbers affects the physiology of normal and cancer cells and whether it could be exploited for cancer therapies has not been investigated. We report that inhibition of NPC formation, a process mostly restricted to proliferating cells, causes selective cancer cell death, prevents tumor growth, and induces tumor regression. Although cancer cells die in response to NPC assembly inhibition, normal cells undergo a reversible cell-cycle arrest that allows them to survive. Mechanistically, reducing NPC numbers results in multiple alterations contributing to cancer cell death, including abnormalities in nuclear transport, catastrophic alterations in gene expression, and the selective accumulation of DNA damage. Our findings uncover the NPC formation process as a novel targetable pathway in cancer cells.Significance:Reducing NPC numbers in cancer cells induces death, prevents tumor growth, and results in tumor regression. Conversely, normal cells undergo a reversible cell-cycle arrest in response to inhibition of NPC assembly. These findings expose the potential of targeting NPC formation in cancer.This article is highlighted in the In This Issue feature, p. 1

In eukaryotic cells, the genetic material is segregated inside the nucleus. This compartmentalization of the genome requires a transport system that allows cells to move molecules across the nuclear envelope, the membrane-based barrier that surrounds the chromosomes. Nuclear pore complexes (NPCs) are the central component of the nuclear transport machinery. These large protein channels penetrate the nuclear envelope, creating a passage between the nucleus and the cytoplasm through which nucleocytoplasmic molecule exchange occurs. NPCs are one of the largest protein assemblies of eukaryotic cells and, in addition to their critical function in nuclear transport, these structures also play key roles in many cellular processes in a transport-independent manner. Here we will review the current knowledge of the NPC structure, the cellular mechanisms that regulate their formation and maintenance, and we will provide a brief description of a variety of processes that NPCs regulate.

Nuclear pore complexes (NPC) are the central mediators of nucleocytoplasmic transport. Increasing evidence shows that many cancer cells have increased numbers of NPCs and become addicted to the nuclear transport machinery. How reducing NPC numbers affects the physiology of normal and cancer cells and whether it could be exploited for cancer therapies has not been investigated. We report that inhibition of NPC formation, a process mostly restricted to proliferating cells, causes selective cancer cell death, prevents tumor growth, and induces tumor regression. Although cancer cells die in response to NPC assembly inhibition, normal cells undergo a reversible cell-cycle arrest that allows them to survive. Mechanistically, reducing NPC numbers results in multiple alterations contributing to cancer cell death, including abnormalities in nuclear transport, catastrophic alterations in gene expression, and the selective accumulation of DNA damage. Our findings uncover the NPC formation process as a novel targetable pathway in cancer cells. Significance: Reducing NPC numbers in cancer cells induces death, prevents tumor growth, and results in tumor regression. Conversely, normal cells undergo a reversible cell-cycle arrest in response to inhibition of NPC assembly. These findings expose the potential of targeting NPC formation in cancer. This article is highlighted in the In This Issue feature, p. 1

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.

This study describes the the role of a nuclear pore complex protein in mammalian in skeletal muscle maintenance, repair, and function., Nuclear pore complexes, the channels connecting the nucleus with the cytoplasm, are built by multiple copies of ∼30 proteins called nucleoporins. Recent evidence has exposed that nucleoporins can play cell type-specific functions. Despite novel discoveries into the cellular functions of nucleoporins, their role in the regulation of mammalian tissue physiology remains mostly unexplored because of a limited number of nucleoporin mouse models. Here we show that ablation of Nup210/Gp210, a nucleoporin previously identified to play a role in myoblast differentiation and Zebrafish muscle maturation, is dispensable for skeletal muscle formation and growth in mice. We found that although primary satellite cells from Nup210 knockout mice can differentiate, these animals show delayed muscle repair after injury. Moreover, Nup210 knockout mice display an increased percentage of centrally nucleated fibers and abnormal fiber type distribution as they age. Muscle function experiments also exposed that Nup210 is required for muscle endurance during voluntary running. Our findings indicate that in mammals, Nup210 is important for the maintenance of skeletal muscle integrity and for proper muscle function providing novel insights into the in vivo roles of nuclear pore complex components.

Nuclear pore complexes are multiprotein channels that span the nuclear envelope, which connects the nucleus to the cytoplasm. In addition to their main role in the regulation of nucleocytoplasmic molecule exchange, it has become evident that nuclear pore complexes and their components also have multiple transport-independent functions. In recent years, an increasing number of studies have reported the involvement of nuclear pore complex components in embryogenesis, cell differentiation and tissue-specific processes. Here, we review the findings that highlight the dynamic nature of nuclear pore complexes and their roles in many cell type-specific functions during development and tissue homeostasis.

Nucleoporins (Nups) are involved in neural development and alterations in Nup genes are linked to human neurological diseases. However, physiological functions of specific Nups and the underlying mechanisms involved in these processes remain elusive. Here we show that tissue-specific depletion of the nucleoporin Seh1 causes dramatic myelination defects in the Central Nervous System (CNS). While proliferation is not altered in Seh1-deficient Oligodendrocyte Progenitor Cells (OPCs), they fail to differentiate into mature oligodendrocytes, which impairs myelin production and remyelination after demyelinating injury. Genome-wide analyses show that Seh1 regulates a core myelinogenic regulatory network and establishes an accessible chromatin landscape. Mechanistically, Seh1 regulates OPCs differentiation by assembling Olig2 and Brd7 into a transcription complex at nuclear periphery. Together, our results reveal that Seh1 is required for oligodendrocyte differentiation and myelination by promoting assembly of an Olig2-dependent transcription complex and define a nucleoporin as key player in the CNS.

Nuclear pore complexes (NPCs), the channels connecting the nucleus with the cytoplasm, are the largest protein structures of the nuclear envelope. In addition to their role in regulating nucleocytoplasmic transport, increasing evidence shows that these multiprotein structures play central roles in the regulation of gene activity. In light of recent discoveries, NPCs are emerging as scaffolds that mediate the regulation of specific gene sets at the nuclear periphery. The function of NPCs as genome organizers and hubs for transcriptional regulation provides additional evidence that the compartmentalization of genes and transcriptional regulators within the nuclear space is an important mechanism of gene expression regulation.

Neutrophils, the front line defenders against infection, express four serine proteases (NSPs) that play roles in the control of cell signaling pathways, defense against pathogens, and whose imbalance leads to pathological conditions. Dissecting the roles of individual NSPs in humans is problematic because neutrophils are end stage cells with a short half life and minimal ongoing protein synthesis. To gain insight into the regulation of NSPs activity we have generated a small molecule chemical toolbox consisting of activity-based probes with different fluorophore detecting groups with minimal wavelength overlap, and highly selective natural and unnatural amino acid recognition sequences. The key feature of these activity-based probes is the ability to use them for simultaneous observation and detection of all four individual NSPs by fluorescence microscopy, a feature never achieved in previous studies. Using these probes we demonstrate uneven distribution of NSPs in neutrophil azurophil granules, such that they seem to be mutually excluded from each other, suggesting the existence of unknown granule targeting mechanisms.

Nuclear pore complexes (NPCs), are large multiprotein channels that penetrate the nuclear envelope connecting the nucleus to the cytoplasm. Accumulating evidence shows that besides their main role in regulating the exchange of molecules between these two compartments, NPCs and their components also play important transport-independent roles, including gene expression regulation, chromatin organization, DNA repair, RNA processing and quality control, and cell cycle control. Here, we will describe the recent findings about the role of these structures in the regulation of gene expression.

In the last decade, the nuclear envelope (NE) has emerged as an important regulator of genome architecture and a central player in gene expression regulation. Nuclear pore complexes (NPCs), the channels that penetrate the NE connecting the nucleus to the cytoplasm, are the largest protein complexes of the NE. Built by multiple copies of roughly 30 different proteins, NPCs were traditionally studied for their role in controlling nucleocytoplasmic transport. But accumulating evidence shows that these massive molecular structures play multiple transport-independent roles that are key for the maintenance of cellular physiology and tissue homeostasis. In this chapter, we will focus on the current knowledge of the role of mammalian NPCs in the regulation of genome organization and gene expression. The recent findings showing that NPCs regulate the activity of specific genes either at the nuclear periphery or inside the nucleus point towards these structures as critical controllers of genome function. Deciphering the molecular mechanism employed by NPCs to modulate specific gene expression programs and to maintain genome integrity are our main challenges for the next decade.

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4+ T cells. Nup210-deficient CD4+ T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic TCR signals and increased levels of Fas, which sensitize Nup210−/− naïve CD4+ T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Caveolin-2 (Cav2) and cJun at the nuclear periphery. While the TCR-dependent and CD4+ T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis; and expose NPCs as key players in the adaptive immune system.

Nuclear pore complexes (NPCs) are multiprotein channels connecting the nucleus with the cytoplasm. NPCs have been shown to have tissue-specific composition, suggesting that their function can be specialized. However, the physiological roles of NPC composition changes and their impacts on cellular processes remain unclear. Here we show that the addition of the Nup210 nucleoporin to NPCs during myoblast differentiation results in assembly of an Mef2C transcriptional complex required for efficient expression of muscle structural genes and microRNAs. We show that this NPC-localized complex is essential for muscle growth, myofiber maturation, and muscle cell survival and that alterations in its activity result in muscle degeneration. Our findings suggest that NPCs regulate the activity of functional gene groups by acting as scaffolds that promote the local assembly of tissue-specific transcription complexes and show how nuclear pore composition changes can be exploited to regulate gene expression at the nuclear periphery.

The study of the Nuclear Pore Complex (NPC), the proteins that compose it (nucleoporins), and the nucleocytoplasmic transport that it controls have revealed an unexpected layer to pathogenic disease onset and progression. Recent advances in the study of the regulation of NPC composition and function suggest that the precise control of this structure is necessary to prevent diseases from arising or progressing. Here we discuss the role of nucleoporins in a diverse set of diseases, many of which directly or indirectly increase in occurrence and severity as we age, and often shorten the human lifespan. NPC biology has been shown to play a direct role in these diseases and therefore in the process of healthy aging.

Maintaining genome integrity is crucial for successful organismal propagation and for cell and tissue homeostasis. Several processes contribute to safeguarding the genomic information of cells. These include accurate replication of genetic information, detection and repair of DNA damage, efficient segregation of chromosomes, protection of chromosome ends, and proper organization of genome architecture. Interestingly, recent evidence shows that nuclear pore complexes, the channels connecting the nucleus with the cytoplasm, play important roles in these processes suggesting that these multiprotein platforms are key regulators of genome integrity.

Suppression of a nuclear pore protein Nup358/RanBP2 is linked to mitotic cell death, but the clinical relevance of this link is unknown. In a recent issue of Cell, Vecchione et al. (2016) show that in approximately 10% of BRAF-like colorectal cancer (CC) patients, Nup358/RanBP2 is critical for survival. Treatment with vinorelbine, a microtubule-depolymerizing drug that inhibits mitosis, might be a potential treatment for these CCs.

Nuclear pore complexes (NPCs) are multiprotein aqueous channels that penetrate the nuclear envelope connecting the nucleus and the cytoplasm. NPCs consist of multiple copies of roughly 30 different proteins known as nucleoporins (NUPs). Due to their essential role in controlling nucleocytoplasmic transport, NPCs have traditionally been considered as structures of ubiquitous composition. The overall structure of the NPC is indeed conserved in all cells, but new evidence suggests that the protein composition of NPCs varies among cell types and tissues. Moreover, mutations in various nucleoporins result in tissue-specific diseases. These findings point towards a heterogeneity in NPC composition and function. This unexpected heterogeneity suggests that cells use a combination of different nucleoporins to assemble NPCs with distinct properties and specialized functions.

Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phosphodiesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites. Fil: D'Angelo, Maximiliano A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Sanguineti, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Reece, Jeffrey M.. National Institute of Environmental Health Sciences; Estados Unidos Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institute of Environmental Health Sciences; Estados Unidos Fil: Torres, Hector Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Flawia, Mirtha Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

We have previously shown that the poly(A) polymerase (PAP) gene of Trypanosoma brucei is interrupted by an intervening sequence. It was postulated that removing this intron by cis-splicing requires a yet unidentified U1 small nuclear RNA (snRNA), which in other organisms engages in base-pair interactions across the 5% splice site during early spliceosome assembly. Here we present a characterization of a 75 nucleotide long candidate T. brucei U1 snRNA. Immunoprecipitation studies indicate that a trimethylguanosine cap structure is present at the 5% end and that the RNA is bound to core proteins common to spliceosomal ribonucleoprotein particles. The U1 snRNA has the potential for extensive intermolecular base pairing with the PAP 5% splice site. We used block replacement mutagenesis to identify sequences necessary for in vivo expression of U1 snRNA. We found that at least two cis-acting elements, tRNA-like A and B boxes, located in the 5%-flanking region are necessary for U1 snRNA synthesis; no internal sequences close to the transcription start site are essential, suggesting a promoter architecture distinct from other trypanosome U-snRNA genes. © 2001 Elsevier Science B.V. All rights reserved.

Nuclear pore complexes are large aqueous channels that penetrate the nuclear envelope, thereby connecting the nuclear interior with the cytoplasm. Until recently, these macromolecular complexes were viewed as static structures, the only function of which was to control the molecular trafficking between the two compartments. It has now become evident that this simplistic scenario is inaccurate and that nuclear pore complexes are highly dynamic multiprotein assemblies involved in diverse cellular processes ranging from the organization of the cytoskeleton to gene expression. In this review, we discuss the most recent developments in the nuclear-pore-complex field, focusing on the assembly, disassembly, maintenance and function of this macromolecular structure.

Nuclear pore complexes are multiprotein channels that span the double lipid bilayer of the nuclear envelope. How new pores are inserted into the intact nuclear envelope of proliferating and differentiating eukaryotic cells is unknown. We found that the Nup107-160 complex was incorporated into assembly sites in the nuclear envelope from both the nucleoplasmic and the cytoplasmic sides. Nuclear pore insertion required the generation of Ran guanosine triphosphate in the nuclear and cytoplasmic compartments. Newly formed nuclear pore complexes did not contain structural components of preexisting pores, suggesting that they can form de novo.

Over the last years it has become evident that the nuclear envelope (NE) is more than a passive membrane barrier that separates the nucleus from the cytoplasm. The NE not only controls the trafficking of macromolecules between the nucleoplasm and the cytosol, but also provides anchoring sites for chromosomes and cytoskeleton to the nuclear periphery. Targeting of chromatin to the NE might actually be part of gene expression regulation in eukaryotes. Mutations in certain NE proteins are associated with a diversity of human diseases, including muscular dystrophy, neuropathy, lipodistrophy, torsion dystonia and the premature aging condition progeria. Despite the importance of the NE for cell division and differentiation, relatively little is known about its biogenesis and its role in human diseases. It is our goal to provide a comprehensive view of the NE and to discuss possible implications of NE-associated changes for gene expression, chromatin organization and signal transduction.

Fil: Kunz, Stefan. University of Bern; Suiza Fil: Beavo, Joseph A.. University of Washington; Estados Unidos Fil: D'angelo, Maximiliano A. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Flawia, Mirtha Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Francis, Sharron H.. Vanderbilt University School of Medicine; Estados Unidos Fil: Johner, Andrea. University of Bern; Suiza Fil: Laxman, Sunil. University of Washington; Estados Unidos Fil: Oberholzer, Michael. University of Bern; Suiza Fil: Rascon, Ana. Universidad Central de Venezuela; Venezuela Fil: Shakur, Yasmin. Otsuka Maryland Medicinal Laboratories; Estados Unidos Fil: Wentzinger, Laurent. University of Bern; Suiza Fil: Zoraghi, Roya. Vanderbilt University School of Medicine; Estados Unidos Fil: Seebeck, Thomas. University of Bern; Suiza

Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.

Interaction between factors from Trypanosoma cruzi extracts and AP-1 sequences was studied by electrophoretic mobility shift assays. Using a double-stranded probe carrying the AP-1 sequence from the SV40 promoter, three specific complexes designated A, B, and C were detected. Complexes A and C were formed when using single-stranded probes. The relative amount of complex B, specific for double-stranded DNA, increased as a function of probe length. Complexes were stabilized by cross-linking with UVC irradiation and resolved on denaturing SDS-PAGE. Complex A generated bands of 60- and 39 kDa; complex B produced two bands of 46- and 43 kDa; and complex C generated one band of 43 kDa. The AP-1 binding activity was much higher in purified nuclear preparations than in soluble fractions, and was detected in crude extracts from the three forms of the parasite. The binding signal, however, was much stronger in amastigote and trypomastigote than in the epimastigote forms. Specific binding was increased by oxidative stress. Antibodies raised against peptides corresponding to conserved domains of mammalian c-Jun and c-Fos detected bands of 40- and 60 kDa, respectively, in a nuclear epimastigote preparation.

Nucleoporins are the constituents of the nuclear pore complex (NPC), the large transport channel that regulates molecular trafficking across the nuclear envelope. Each mammalian NPC is an ~60–125 MDa multiprotein assembly built from multiple copies of ~30 different nucleoporins.1 In addition to their role in nucleocytoplasmic transport, the NPC and its components have been implicated in a variety of other cellular processes, ranging from gene expression regulation to cytoskeleton organization. Importantly, some of these roles have been attributed to nucleoporins located outside of the NPC, indicating that they can function independently of this structure. In higher eukaryotes, the nuclear envelope breaks down during prometaphase, causing NPCs to disassemble into nucleoporin subcomplexes. These complexes are then reused to build NPCs in the newly formed nuclear envelopes. But a growing body of evidence indicates that nucleoporins do not just sit in the mitotic cytoplasm waiting to be reassembled into NPCs; in fact, they act as critical regulators of a number of mitotic events that ensure high-fidelity chromosome segregation, and thereby prevent aneuploidy. These include formation of a bipolar mitotic spindle; establishment of stable kinetochore-microtubule attachments; kinetochore assembly; timely chromosome segregation; and correct localization of spindle assembly checkpoint proteins.1 Although depletion or overexpression of a number of nucleoporins causes multipolar spindles,2-4 a phenotype frequently associated with supernumerary centrosomes, whether nucleoporins actively participate in centrosome biogenesis was unknown. Now, work from the Wong group highlights a critical role for nucleoporin Nup62 in this process.5 The centrosome is a cytoplasmic, non-membranous organelle composed of 2 orthogonally arranged barrel-shaped structures, the centrioles, each encircled by 9 triplet microtubules and an electron-dense matrix in which the centrioles are embedded, termed pericentriolar material (PCM).6 As the major microtubule-organizing center in animal cells, the centrosome nucleates and anchors the interphase and mitotic microtubule arrays. The centrosome also contributes to cytokinesis and subsequent cell cycle progression, as well as to spindle positioning.6 Centrosomes divide once per cell cycle, in a mechanism that is intimately connected to centriole duplication. A G1 cell typically contains one pair of centrioles, which duplicate in S phase giving rise to 2 pairs. The 2 pairs of centrioles remain together through G2, where they recruit additional PCM proteins in a process called centrosome maturation. During prophase, they start migrating to opposite poles to assemble the mitotic spindle. Using an siRNA-mediated approach to reduce Nup62 expression in HeLa cells, Hashizume and Moyori observed a significant increase in the number of multinucleated cells, which was associated with a cell cycle arrest at the G2/M phase and increased cell death.5 Electron microscopy analysis of Nup62-depleted cells showed normal NPC morphology, leading the authors to propose an NPC-independent role for this nucleoporin in cell cycle regulation. Strikingly, Nup62-depleted interphase cells displayed aberrant centrosomes, consisting of 3 centrioles, instead of the pair characteristic of normal cells, suggesting centriole duplication defects. Moreover, Nup62 depletion induced centrosome amplification, multipolar spindle formation, improper spindle orientation, and alterations in microtubule polymerization.5 But how does Nup62 regulate centrosome homeostasis? The authors found that Nup62 localized to centrosomes/spindle poles via its C-terminal domain, where it associated with the centrosomal proteins gamma-tubulin and hSAS-6, and that its depletion caused mislocalization of several centrosomal components. This suggests that Nup62 is involved in the recruitment of critical proteins to the centrosome. Altogether, these results provide the first compelling evidence for a nucleoporin as a key regulator of centrosome biogenesis and maturation.5 An important task for the future is to define the detailed molecular mechanism through which Nup62 exerts these effects. As suggested by the authors, Nup62 may be acting through its interacting partner SAS-6, a protein required for centrosome duplication.7 It will also be interesting to determine if other members of the Nup62 complex (Nup45, Nup54, and Nup58) are centrosomal and/or involved in regulating centrosome homeostasis. In C. elegans, spindle orientation defects were found after depletion of the Nup62 complex homologs,8 suggesting that these proteins play mitotic roles. Finally, supernumerary centrosomes and multipolar spindles may also arise from perturbations of microtubule dynamics, and it would be exciting to investigate whether Nup62 is involved in this process.

SummaryNuclear pore complexes (NPCs) are built from ∼30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination.

SummaryIn dividing cells, nuclear pore complexes (NPCs) disassemble during mitosis and reassemble into the newly forming nuclei. However, the fate of nuclear pores in postmitotic cells is unknown. Here, we show that NPCs, unlike other nuclear structures, do not turn over in differentiated cells. While a subset of NPC components, like Nup153 and Nup50, are continuously exchanged, scaffold nucleoporins, like the Nup107/160 complex, are extremely long-lived and remain incorporated in the nuclear membrane during the entire cellular life span. Besides the lack of nucleoporin expression and NPC turnover, we discovered an age-related deterioration of NPCs, leading to an increase in nuclear permeability and the leaking of cytoplasmic proteins into the nucleus. Our finding that nuclear “leakiness” is dramatically accelerated during aging and that a subset of nucleoporins is oxidatively damaged in old cells suggests that the accumulation of damage at the NPC might be a crucial aging event.