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PubMed2001

Identification of a 60S preribosomal particle that is closely linked to nuclear export.

A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.

Active Transport, Cell Nucleus↗
PubMed2005

Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.

The nuclear membrane is a tight barrier against the delivery of therapeutic genes into non-dividing tissue cells. Overcoming this barrier with the aid of peptidic nuclear localization signals (NLS) is crucial for improving the performance of synthetic gene-delivery vehicles. In this article, we examine the nuclear transport of lambda phage particles displaying various peptides containing the minimum NLS of SV40 T antigen on their surface. As the minimum NLS (PKKKRKV) is a binding domain to importin alpha, recombinant proteins and molecular conjugates containing this peptide accumulate into the nucleus efficiently. However, we find that the C-terminal and N-terminal structures besides the minimum NLS profoundly affect the efficiency of the nuclear transport of the phage particles as well as their binding capacity to importin alpha: either truncation of a few amino acid residues from the C-terminus or the replacement of the N-terminus with a FLAG- or c-myc-tag abolish both of these biological activities. The structure of the optimized NLS is unpredictable from conventional protein transport assay and from the structural analysis in silico. Our results reveal that the objects with 50 nm in diameter can pass through the nuclear pore complex when the optimized NLS is displayed at a sufficient density on their surface.

Active Transport, Cell Nucleus↗
PubMed1997

Nucleocytoplasmic transport: signals, mechanisms and regulation.

In eukaryotic organisms, DNA replication and RNA biogenesis occur in the cell nucleus, whereas protein synthesis occurs in the cytoplasm. Integration of these activities depends on selective transport of proteins and ribonucleoprotein particles between the two compartments. Transport across the nuclear envelope occurs through large multiprotein structures, termed nuclear pore complexes. It is signal-mediated and requires both energy and soluble factors, including shuttling carriers. Here I summarize current understanding of nucleocytoplasmic transport and illustrate the importance of regulated transport for signal transduction.

Amino Acid Sequence↗
PubMed2000

Autoimmune tests in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by an immune mediated, irreversible destruction of the small intrahepatic bile ducts leading to progressive liver cirrhosis and frequently to liver failure. The course of the disease is variable and an early diagnosis is desirable to identify individuals with rapidly progressing disease, to initiate adequate therapeutic measures and to evaluate the necessity of liver transplantation. Serological tests represent the single most important diagnostic feature of PBC because liver histology, biochemistry, or clinical syndrome alone are not reliable in this respect. The molecular definition of the autoantigen targets of antimitochondrial antibodies (AMA) has resulted in the development of reproducible and effective serological testing strategies. AMA directed against the ketoacid dehydrogenase complex are highly disease-specific but not directed against liver-specific target structures. Despite a high disease specificity, their usefulness for predicting the course of disease, the timing of liver transplantation, or disease recurrence after transplantation is limited. The realization that about 5% of patients with PBC do not display AMA has led to the identification of PBC-specific antinuclear autoantibodies directed against the nuclear pore complex and other targets. The overlap of PBC with autoimmune hepatitis and primary sclerosing cholangitis represents a diagnostic challenge in which autoantibody determinations play a central role and contribute to the administration of suitable treatment options.

Antibodies, Antinuclear↗
PubMed1995

Distinct phosphorylation sites differentially influence facilitated transport of an NLS-protein and its subsequent intranuclear binding.

We measured the nuclear transport of radiolabeled fusion proteins consisting of variants of the Simian Virus 40 large T antigen's nuclear localization sequence region linked to beta-galactosidase, itself a cytoplasmic protein. We microinjected the fusion protein variants into the cytoplasm of living Xenopus oocytes or supplied them to the surface of oil-isolated oocyte nuclei via paired beads or cytoplasm. Presence of the cdc2 kinase site (124T) on the amino flank of the nuclear localization sequence (126PKKKRKV132) greatly enhances facilitated transport through the nuclear pore complex; additional presence of the casein kinase II site (112S) enhances subsequent intranuclear binding.

Amino Acid Sequence↗
PubMed2000

The Ct-RAE1 protein interacts with Balbiani ring RNP particles at the nuclear pore.

RAE1 is an evolutionarily conserved protein that associates with both mRNPs and nucleoporins, and may bridge the interaction between mRNP export cargoes and the nuclear pore complex (NPC). However, the mechanism by which RAE1 functions in mRNA export is still unknown and the time point at which RAE1 interacts with the exported RNP has not been directly investigated. Here we have addressed this question in the Balbiani ring (BR) system of Chironomus tentans using immunoelectron microscopy. The RAE1 protein of C. tentans, Ct-RAE1, is 70% identical to human RAE1/mrnp41 (hRAE1) and is recognized by antibodies raised against hRAE1. As in vertebrate cells, Ct-RAE1 is concentrated at the nuclear envelope and also dispersed throughout the nuclear interior. Here we show that Ct-RAE1 does not bind to the BR particle either cotranscriptionally or in the nucleoplasm. Instead, the interaction between Ct-RAE1 and the exported BR particle occurs at the NPC. Moreover, the localization of Ct-RAE1 at the NPC is correlated with the presence of an exported RNP in the NPC. Finally, the anti-RAE1 antibody does not label the cytoplasmic side of BR particles in transit through the central channel, which indicates that Ct-RAE1 either remains anchored at the nuclear side of the NPC during translocation of the RNP through the central channel or becomes transiently associated with the RNP but is rapidly released into the cytoplasm.

Amino Acid Sequence↗
PubMed1992

High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores.

The nuclear envelope (NE) of amphibian oocytes can be readily isolated in relatively structurally intact and pure form and has been used extensively for structural studies. Using high resolution scanning electron microscopy (HRSEM), both surfaces of the NE can be visualized in detail. Here, we demonstrate the use of HRSEM to obtain high resolution information of NE structure, confirming previous data and providing some new information. NEs, manually isolated from Triturus cristatus oocytes, have been mounted on conductive silicon chips, fixed, critical point dried and coated with a thin, continuous film of chromium or tantalum and viewed at relatively high accelerating voltage in a field emission scanning electron microscope with the sample within the objective lens. Both nucleoplasmic and cytoplasmic surfaces of the nuclear pore complexes (NPC) have been visualized, revealing the cytoplasmic coaxial ring, associated particles, central plug/transporter and spokes. The nucleoplasmic face is dominated by the previously described basketlike structure attached to the nucleoplasmic coaxial ring. In Triturus, a novel, highly regular flat sheet of fibers, termed the NE lattice (NEL) has been observed attached to the distal ring of the NPC basket. The NEL appears to be distinct from the nuclear lamina. Evidence for the NEL is also presented in thin TEM sections from Triturus oocytes and GVs and in spread NEs from Xenopus. A model is presented for NEL structure and its interaction with the NPCs is discussed.

Amphibians↗
PubMed1997

v-erbA oncogene initiates ultrastructural changes characteristic of early and intermediate events of meiotic maturation in Xenopus oocytes.

The growth-promoting properties of the retroviral v-erbA oncogene, a highly mutated version of the chicken thyroid hormone receptor (TR) alpha, have so far exclusively been linked to dominant repression of the antimitogenic roles of TR and retinoic acid receptors. Here we show that when expressed in Xenopus oocytes v-ErbA induced ultrastructural changes characteristic of early and intermediate events of meiotic maturation by activating gene transcription. v-ErbA-induced maturation events occurred without activation of the cAMP/maturation-promoting factor signal pathway and were arrested prior to meiotic spindle formation. The effects of v-ErbA were not mimicked by a dominant negative in vitro-generated mutant of human TR, suggesting that v-ErbA can contribute to cell cycle reentry by interference with regulatory pathways distinct from those involving TR. Interestingly, a portion of v-ErbA expressed in oocytes was present at the cytoplasmic fibrils of the nuclear pore complexes, suggesting that in addition to its intranuclear function v-ErbA may modulate nucleocytoplasmic transport.

Animals↗
PubMed1996

Accessing nuclear structure for field emission, in lens, scanning electron microscopy (FEISEM).

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding, its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces.

Animals↗
PubMed2000

Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

Cell Compartmentation↗
PubMedDate not supplied

Current concepts in nuclear pore electrophysiology.

Over 4 decades ago, microelectrode studies of in situ nuclei showed that, under certain conditions, the nuclear envelope (NE) behaves as a barrier opposing the nucleocytoplasmic flow of physiological ions. As the nuclear pore complexes (NPCs) of the NE are the only pathways for direct nucleocytoplasmic flow, those experiments implied that the NPCs are capable of restricting ion flow. These early studies validated electrophysiology as a useful approach to quantify some of the mechanisms by which NPCs mediate gene activity and expression. Since electron microscopy (EM) and other non-electrophysiological investigations, showed that the NPC lumen is a nanochannel, the opinion prevailed that the NPC could not oppose the flow of ions and, therefore, that electrophysiological observations resulted from technical artifacts. Consequently, the initial enthusiasm with nuclear electrophysiology faded out in less than a decade. In 1990, nuclear electrophysiology was revisited with patch-clamp, the most powerful electrophysiological technique to date. Patch-clamp has consistently demonstrated that the NE has intrinsic ion channel activity. Direct demonstrations of the NPC on-off ion channel gating behavior were published for artificial conditions in 1995 and for intact living nuclei in 2002. This on-off switching/gating behavior can be interpreted in terms of a metastable energy barrier. In the hope of advancing nuclear electrophysiology, and to complement the other papers contained in this special issue of the journal, here I review some of the main technical, experimental, and theoretical issues of the field, with special focus on NPCs.

Animals↗
PubMed2006

Nucleocytoplasmic transport of plasmid DNA: a perilous journey from the cytoplasm to the nucleus.

Nonviral vectors represent a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Before synthetic vector systems can be used for clinical applications, their limited efficacy must be addressed. At the cellular level, successful gene transfer is dependent on several additional factors including DNA uptake, release from the DNA-vector complex, and nucleocytoplasmic transport. This paper reviews the major metabolic and physical impediments that plasmid DNA vectorized by synthetic vectors encounters between the cytosol and the nucleus. Plasmid DNA that escapes the endolysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing the number of intact plasmids that reach the nuclear envelope. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope during cell division or active nuclear transport via the nuclear pore complex. In the nucleus, plasmid DNA is relatively stable, but its transcription and its fate during cell division are still debated. A better understanding of the cellular and molecular basis of nonviral gene transfer during nucleocytoplasmic trafficking may provide strategies to overcome those obstacles that limit the efficiency of nonviral gene delivery. We review some of the current methods of gene transfer mediated by synthetic vectors, highlighting systems that exploit our actual knowledge of the nucleocytoplasmic transport of plasmid DNA.

Biological Transport↗
PubMed2000

Nuclear dynamics in Arabidopsis thaliana.

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to beta-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.

Actin Cytoskeleton↗
PubMed2000

Interdependence between a 55 kDa protein (p55) and a 12 kDa protein (p12) in facilitating the nuclear entry of goat uterine estrogen receptor under cell-free conditions.

A 55 kDa nuclear localization signal binding protein (p55) is involved in the transport of the goat uterine estrogen receptor from the cytoplasm to the nuclear pore complex (NPC). p55 forms a complex with a 12 kDa protein (p12) which in turn becomes 'docked' at the NPC. The present study reports on the purification and functional characterization of p12. Both p55 and p12 are Mg2+-dependent ATPases. The protein-protein interactions that take place between these two molecules at the NPC cause an enhancement in the net ATPase activity associated with the protein complex. Presumably, this enhanced ATPase function helps in the final nuclear entry of the estrogen receptor; p55 remains associated with p12 at the nuclear entry site under these conditions.

Adenosine Triphosphate↗
PubMed1996

Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids.

Nuclear proteins contain information within their primary structures which causes them to accumulate selectively in the nucleus [1,2] by associating with the cytosolic receptor importin [3]. The alpha subunit of importin binds the nuclear localization signal (NLS), and the beta subunit docks at the nuclear pore complex. The NLS of the simian virus 40 large T-antigen (SV40 T-ag) is a single cluster of basic amino acids (PKKKRKV132; single-letter code, the basic amino acids are shown in bold; [4,5]), whereas the NLS of nucleoplasmin is bipartite. The nucleoplasmin NLS requires two essential clusters of basic amino acids, separated by a mutation-tolerant spacer (KRPAATKKAGQAKKKK171; [6] [7]). A SwissProt database search shows that more than 50% of nuclear proteins contain a match to this consensus, and many NLSs have since been found to conform to this type of motif in yeast, plants and animals [8-10]. A different NLS (PAAKRVKLD) has been reported in the oncoprotein c-Myc, but it has received little attention because, unlike other known NLSs, only three of nine residues are basic [11], and one residue is even acidic. Here, we report that constructs containing an inactive basic cluster downstream of the bipartite signal of nucleoplasmin can be directed to the nucleus by flanking them with specific neutral and acidic residues taken from the signal reported for c-Myc. Nuclear targeting by the single cluster KKKK is dependent on it being preceded by PAA and is stimulated if it is followed by the dipeptide LD. The relative positions of these elements are crucial to the function of these NLSs. All regions of the unconventional signal of c-Myc are functionally important. Contrary to conventional views, neutral and even acidic amino acids can play crucial roles in NLSs.

Amino Acid Sequence↗
PubMed2003

Actin-filled nuclear invaginations indicate degree of cell de-differentiation.

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.

Actin Cytoskeleton↗
PubMed1999

Cellular uptake and nuclear delivery of recombinant adenovirus penton base.

An Ad2 capsid component, the penton base, expressed as recombinant protein, was found to be capable of affecting the entire entry pathway of adenovirion in HeLa cells, i.e., cell attachment, endocytosis, vesicular escape, intracytoplasmic movement, and translocation through the nuclear pore complex. Data with pentamerization-defective mutants suggested that none of these successive steps depended upon penton base pentamer status, indicating that the peptide domains responsible for these functions were carried by the monomer. Observations performed with wild-type (WT) and an integrin-binding-site double-mutant (K288E340) suggested that the penton base could enter the cell via an alternative, RGD- and LDV-independent, pathway. Of three mutants that were found to be defective in nuclear addressing in insect cells, only one, W165H, was also altered in nuclear transport in HeLa cells. The other two, W119H and RRR547EQQ, showed a WT pattern of nuclear localization in HeLa cells, suggesting that the region including tryptophan-119 and the basic signal at position 547 did not act as a nuclear localization signal in the human cell context. The integrity of cellular structures and the cytoskeleton seemed to be required for the vectorial movement and nuclear import of WT penton base, as suggested by experiments using permeabilized HeLa cells, isolated nuclear membranes, and cytoskeleton-targeted drugs.

Adenoviruses, Human↗
PubMed1996

Nuclear pores and macromolecular assemblies involved in nucleocytoplasmic transport.

Transport in and out of the nucleus is mediated by nuclear pore complexes (NPCs). Information has recently been emerging both on the structure of many of the proteins involved in this transport and on the complexes formed between them. In addition to NPC-based complexes, such as those based on nucleoporins Nsp1p and p62, cytoplasmic protein macromolecular assemblies are also important for nucleocytoplasmic transport.

Animals↗