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PubMed1998

Nucleocytoplasmic shuttling by protein nuclear import factors.

Protein nuclear import factors are not, in general, believed to function in the nuclear export of macromolecules and their reutilization therefore requires their recycling from the nucleus to the cytoplasm. Two possible mechanisms for recycling have been proposed. On the one hand, protein import factors such as importin beta and transportin (Trn) could continuously shuttle between cytoplasm and nucleoplasm. On the other hand, these proteins could penetrate into the nucleus only as far as the inner surface of the nuclear pore complex and then directly return to the cytoplasm. In this manuscript, we have used microinjection analysis in human cells, and in vitro nuclear assays, to demonstrate that importin beta, transportin and importin alpha are all nucleocytoplasmic shuttle proteins that efficiently enter and exit the cell nucleoplasm. In the case of transportin, we have mapped sequences required for nucleocytoplasmic shuttling to the carboxy-terminal 270 amino acids of this 890 amino acid import factor, thus demonstrating that nuclear export is independent of the amino-terminal Ran-binding domain of Trn. We further show that Trn shuttling is independent of nuclear RNA transcription. Overall, these data suggest that nucleocytoplasmic shuttling is likely to be a general attribute of protein nuclear import factors.

Binding Sites↗
PubMed2001

Importin 13: a novel mediator of nuclear import and export.

Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO-1/sentrin-conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin beta-like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Cell Nucleus↗
PubMed1995

Diffusion across the nuclear envelope inhibited by depletion of the nuclear Ca2+ store.

Intact, isolated nuclei and a nuclear membrane (ghost) preparation were used to study regulation of the movement of small molecules across the Xenopus laevis oocyte nuclear membrane. In contrast to models of the nuclear pore complex, which assume passive bidirectional diffusion of molecules less than 70 kilodaltons, diffusion of intermediate-sized molecules was regulated by the nuclear envelope calcium stores. After depletion of nuclear store calcium by inositol 1,4,5-trisphosphate or calcium chelators, fluorescent molecules conjugated to 10-kilodalton dextran were unable to enter the nucleus. Dye exclusion after calcium store depletion was not dependent on the nuclear matrix because it occurred in nuclear ghosts lacking nucleoplasm. Smaller molecules and ions (500-dalton Lucifer yellow and manganese) diffused freely into the core of the nuclear ghosts and intact nuclei even after calcium store depletion. Thus, depletion of the nuclear calcium store blocks diffusion of intermediate-sized molecules.

Adenosine Triphosphate↗
PubMed1990

EM visualization of nucleocytoplasmic transport processes.

The nuclear envelope is strategically located between the nucleoplasm and cytoplasm, and, as such, can play a major role in controlling cellular activity by regulating the exchange of macromolecules between these two compartments. The nuclear pore complexes, which are located within circular areas formed by fusion of the inner and outer membranes of the envelope, represent the primary, if not the exclusive, exchange sites. Individual pores are able to function in both protein import and RNA efflux from the nucleus. Translocation of macromolecules occurs by either passive diffusion or facilitated transport through central channels within the pores. The functional size of the diffusion channel is approximately 9 to over 12 nm in diameter depending on the cell type. The width of the transport channel varies as a function of the number and effectiveness of the specific nuclear targeting signals contained within the permeant molecule. The maximum diameter of the channel can be over 26 nm. Nucleocytoplasmic exchanges can be regulated either by (1) differences in the properties of the transported molecule (molecular size and signal content) or (2) changes in the properties of the pore complexes, which can effect both diffusion and transport.

Amino Acid Sequence↗
PubMed2006

Mechanism of PNA transport to the nuclear compartment.

We evaluated the nuclear uptake of fluorescently labeled peptide nucleic acids and measured the binding of unlabeled peptide nucleic acids (PNAs) to the endogenous HER-2/neu promotor in digitonin-permeabilized SK-BR-3 cells. Fluorescently labeled PNAs readily enter the nucleus of digitonin-permeabilized cells, and binding to the chromosomal target sequence was detected with a bis-PNA. Nuclear uptake and target sequence binding were inhibited by N-ethylmaleimide (NEM) and GTPgammaS. We conclude that PNAs are transported into the nucleus through an energy-dependent process involving the nuclear pore complex.

Active Transport, Cell Nucleus↗
PubMed1988

Paired helical filaments and the cytoplasmic-nuclear interface in Alzheimer's disease.

The cytoplasmic-nuclear interface has been investigated by conventional thin sectioning electron microscopy of neurons from frontal lobe biopsies of 13 patients with Alzheimer's disease. Nine patients were in the early and intermediate stages of the disease and four patients in the advanced stage. Fascicles of paired helical filament-like strands and paired helical filaments appose the nuclear envelope, the nuclear pore complexes and the perinuclear polysomes. Paired helical filament profiles have also been identified in the nucleoplasm. These observations indicate that the cytoplasmic-nuclear interface and, consequently, the relationships between the cytoplasm and the nucleus, might be impaired in Alzheimer's disease.

Alzheimer Disease↗
PubMed1978

The ultrastructure of non-membranous nuclear ghosts.

Non-membranous HeLa cell nuclear ghosts, representing non-membranous nuclear envelope or 'skeletal' components, have been examined in whole-mount fashion by transmission electron microscopy. Major components of the ghosts include annuli with inner and outer diameters of 43 and 90 nm, respectively, which are consistent in dimensions with nuclear pore complexes. Also present are rod-like images (260 nm in length and 50 nm in width or diameter) representing either previously unobserved nuclear structures, or condensations of repeating functional units not otherwise observable. The annular and rod-like images were also observed when various steps in the ghost isolation procedure, such as the use of detergents, 0.5 M MgCl2 and polylysine attachment of the ghosts to electron-microscope grids, were circumvented. The annular and rod-like images are connected into linear and polygonal arrays by strands (15-30 nm in width) that are sensitive to DNase I and DNase II but resistant to nuclease S1. Thus, although the non-membranous ghosts from HeLa cells are composed primarily of protein, enzymic dissection indicates that their gross integrity is markedly dependent on double-stranded DNA. Nuclear ghosts prepared from a wide range of species including mammals, birds and plants, exhibited essentially the same components and organization.

Cell Nucleus↗
PubMed2006

Synthetic peptides as non-viral DNA vectors.

The use of multiple peptide motifs to provide effective gene delivery holds great promise as an elegant, non-immunogenic approach to gene therapy. The molecular understanding of cell and viral biology provides a strong foundation on which to pursue this objective. Synthetic peptides containing multiple lysines and/or arginines (occasionally ornithines) provide natural polycations for multivalent electrostatic binding of DNA, and for DNA compaction into particles suitable for gene delivery. These cationic peptides can incorporate additional functional motifs (e.g. for translocating DNA into the nucleus) and they can be linked by disulphide bonds to produce high molecular reducible polycations with superior properties for gene therapy. Many factors influence the size, surface charge and stability of peptide/DNA particles. For in vivo use, uncharged particles resistant to disruption by salt and protein, and targeted to tissue-specific membrane molecules, will be required. Entry into the cell is via one of the endocytic pathways, depending on particle size and (in principle) the target cell surface molecule. Peptide motifs for endocytic escape are based mainly on the anionic fusogenic peptide of influenza virus haemagglutinin and on histidine-rich peptides (where the buffering properties of the imidazole group cause osmotic swelling and probably rupture of endocytic vesicles). Once in the cytosol, translocation of DNA plasmids across the nuclear pore complex into the nucleus is a crucial step, because most target cells for gene therapy are either non-dividing or slowly dividing. Nuclear translocation can be achieved by classical nuclear localising motifs, or more simply by (Lys)16 and other cationic peptides.

Amino Acid Sequence↗
PubMed2001

Two closely related human nuclear export factors utilize entirely distinct export pathways.

Nuclear mRNA export mediated by the human protein TAP requires a carboxy-terminal domain that directly interacts with components of the nuclear pore complex. Here we demonstrate that NXF3, a human RNA binding protein related to TAP, lacks this domain yet retains the ability to export tethered RNA transcripts and to shuttle between the nucleus and the cytoplasm. NXF3 contains a novel Crm1-dependent nuclear export signal that compensates in cis for the loss of the nuclear pore targeting domain. NXF3-dependent RNA export is therefore blocked by Crm1-specific inhibitors that do not affect TAP function. Thus, while the related TAP and NXF3 proteins are both capable of mediating nuclear RNA export, they do so via unrelated export pathways.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

O-GlcNAc glycosylation: a signal for the nuclear transport of cytosolic proteins?

Year 2004 marks the 20th anniversary of the discovery of O-linked N-acetylglucosamine (O-GlcNAc) by Gerald W. Hart. Despite interest for O-GlcNAc, the functions played by this single monosaccharide remain poorly understood, though numerous roles have been suggested, among which is the involvement of O-GlcNAc in the nuclear transport of cytosolic proteins. This idea was first sustained by studies on bovine serum albumin that showed that the protein could be actively carried to the nucleus when it was modified with sugars. In this paper, we will review data on this puzzling problem. We will first describe the well-established nuclear localisation signal (NLS)-dependent nuclear transport by presenting the different factors involved, and then, we will examine where and how O-GlcNAc could be involved in nuclear transport. Whereas it has been suggested that O-GlcNAc could interfere at two levels in the nuclear transport both by modifying proteins to be translocated to the nucleus and by modifying the nucleoporins of the nuclear pore complex, according to us, this second idea seems unlikely. Part of this study will also be dedicated to a relatively new concept in the nuclear transport: the role of the 70-kDa heat shock proteins (HSP70). The action of the chaperone in nuclear translocation was put forward 10 years ago, but new findings suggest that this mechanism could be linked to O-GlcNAc glycosylation.

Acetylglucosamine↗
PubMed2004

Nuclear transport erupts on the slopes of Mount Etna.

Nuclear pore complexes (NPCs) mediate the active transport of large substrates and allow the passive diffusion of small molecules into the nucleus of eukaryotic cells. The EMBO Workshop on the Mechanisms of Nuclear Transport focused on NPCs and on the soluble nucleocytoplasmic transport machinery. This meeting, organized by Valérie Doye (Institut Curie, Paris) and Ed Hurt (University of Heidelberg), was held within view of Mount Etna at Taormina, Sicily (November 1-5, 2003). Presentations emphasized the dynamic properties of the nuclear trafficking machinery, and demonstrated the continuity of nuclear transport with processes in the nucleus and cytoplasm.

Active Transport, Cell Nucleus↗
PubMed1979

Active movements of the chromatoid body. A possible transport mechanism for haploid gene products.

Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.

Animals↗
PubMed1999

Nuclear import and export pathways.

Macromolecules enter or leave the nucleus by using nuclear localization signals (NLS), or nuclear export signals (NES), respectively. Different types of NLS and NES are recognized directly or indirectly via adapters, by transport receptors. All transport receptors identified thus far are members of the same family and share an ability to shuttle between the nucleus and the cytoplasm, and to interact with the small GTPase Ran and with nucleoporins at the nuclear pore complex (NPC). The GTPase Ran regulates the interaction of transport receptors with either cargoes, or adapters, or nucleoporins and is crucial in providing directionality to nuclear import and export. Surprisingly, GTP hydrolysis by Ran is not required for translocation of some receptor/cargo complexes through the NPC. One of the challenges for the future will be to establish the mechanisms of translocation through the NPC of different transport receptors together with their cargoes. J. Cell. Biochem. Suppls. 32/33:76-83, 1999.

Animals↗
PubMed1997

Ionophore-releasable lumenal Ca2+ stores are not required for nuclear envelope assembly or nuclear protein import in Xenopus egg extracts.

The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca2+ stores via inositol 1,4,5-trisphosphate-gated Ca2+ channels is required for nuclear assembly in Xenopus egg extracts. Other work suggested that lumenal Ca2+ stores are required for nuclear protein import in mammalian cells in vivo, but not in vitro. Here, we rigorously tested the role of lumenal Ca2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca2+ stores were depleted by pretreating the extracts with Ca2+ ionophores (ionomycin, A23187) or inhibitors of Ca(2+)-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca(2+)-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca2+ stores confirms a previous unpublished observation by Greber and Gerace, and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca2+. Finally, we concluded that, contrary to our expectations, lumenal Ca2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.

Animals↗
PubMed2001

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization.

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.

Animals↗
PubMed2005

The view from Awaji island: past, present, and future of RCC1 and the Ran GTPase system.

The International Symposium on Ran and the Cell Cycle was held on October 1-4, 2005, at the Awaji Island Resort near Osaka, to celebrate the career and scientific achievements of Professor Takeharu Nishimoto. One hundred of his former lab members, collaborators and other scientific colleagues from around the world attended the symposium organized by Mary Dasso (National Institutes of Health) and Yoshihiro Yoneda (Osaka University). The program was divided into sessions on cell cycle and chromosomes, nuclear import and export of proteins and RNA, nuclear envelope and the nuclear pore complex, and RCC1 and chromatin. Dr. Nishimoto's retirement from Kyushu University is a perfect time to look back at the history of Ran and RCC1, assess the current state of the field, and discuss the challenges that remain in order to unravel the complexities of the Ran GTPase system.

Biological Transport↗
PubMed1993

Assembly of envelope structure with vesicles associated with Ku-homologous protein in Xenopus egg extract in the absence of chromatin.

To study the process of nuclear envelope assembly at the end of mitosis, we developed a chromatin-free in vitro system for assembly of envelope structures in Xenopus interphase egg extract, and examined the participation of Ku-homologous protein in the assembly. The envelope structure assembled spontaneously in the absence of chromatin or DNA between glass plates under a condition that minimized generation of flow of the extract. Morphological study using an electron microscopy has revealed that the membrane surrounding the envelope structure is a double membrane that contains gaps resembling nuclear pore complex. Their assembly was dependent on ATP and was inhibited by the addition of GTP-gamma-S or N-ethylmaleimide. Depletion of a pre-nuclear vesicle by preincubating the interphase egg extracts with large excess of sperm head chromatins impaired the assembly. The membrane vesicle, which was associated with Ku-homologous protein of Xenopus, participated in the assembly as proven by reaction with monoclonal antibody made specific for Ku p70 protein. However, the assembly process of the envelope structure was inhibited only slightly by the antibody, suggesting that the Ku-homologous protein does not participate in the fusion process of vesicles to form the envelope structure.

Adenosine Triphosphate↗