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PubMed1997

Evidence for covalent modification of the nuclear dot-associated proteins PML and Sp100 by PIC1/SUMO-1.

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1-specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.

Animals↗
PubMed1998

Identification of a tRNA-specific nuclear export receptor.

In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.

Amino Acid Sequence↗
PubMed1996

Cytoplasmic annulate lamellae in cultured cells: composition, distribution, and mitotic behavior.

We have used antibodies against different proteins of the nuclear pore complex to identify cytoplasmic annulate lamellae by immunocytochemistry at the light- and electron-microscopic level in various lines of cultured mammalian cells. Although annulate lamellae (AL) are seldom observed in some cell lines, they occur in large numbers in other lineages, also with cell-type-specific size distributions. AL are especially abundant and prominent in African green monkey kidney epithelial cells of line RC37 and bovine mammary gland epithelial, cells of line BMGE. We have studied the distribution of AL in relation to other organelles by using double-label immunofluorescence microscopy and have demonstrated a significant relationship between AL and rough endoplasmic reticulum. We have further shown that AL are disassembled during prophase and reassembled at the end of mitosis, almost concomitantly with the disassembly and reassembly of the nuclear envelope. Cultured mammalian cells that are rich in AL can therefore be used as suitable models for studies of the biogenesis of these lamellae, which can conveniently be detected by immunofluorescence microscopy.

Animals↗
PubMed1991

A strategy for the reconstruction of structures possessing axial symmetry: sectioned axonemes in sperm flagella.

The images of complex biological structures seen in the electron microscope, possessing an n-fold rotational symmetry, can be enhanced by averaging the axially repeating motif in order to improve their signal-to-noise ratio; this requires that the slices with n-fold symmetry do not exhibit distortions relative to one another. A strategy is proposed to detect the relative distortions and to remove them in order to obtain reliable results from the averaging process. Extensive use is made of cross-correlation analysis and of interpolation; because the procedure involves iterated resampling of the image, it is essential to adopt interpolation algorithms which preserve the spectral power. The procedure is illustrated by the analysis of transverse sections of the sperm flagellum axoneme of a stick insect; it might be used for the reconstruction of microtubules, nuclear pore complexes, virus capsids, and other supramolecular aggregates. The reconstructed images of axoneme sections reveal new information about the interactions between adjacent doublets; in particular, a double conjunction connecting the inner dynein arm with the nearest B-tubule has been consistently observed.

Animals↗
PubMed2002

GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta.

The interaction between nuclear pore proteins (nucleoporins) and transport factors is crucial for the translocation of macromolecules through nuclear pores. Many nucleoporins contain FG sequence repeats, and previous studies have demonstrated interactions between repeats containing FxFG or GLFG cores and transport factors. The crystal structure of residues 1-442 of importin-beta bound to a GLFG peptide indicates that this repeat core binds to the same primary site as FxFG cores. Importin-beta-I178D shows reduced binding to both FxFG and GLFG repeats, consistent with both binding to an overlapping site in the hydrophobic groove between the A-helices of HEAT repeats 5 and 6. Moreover, FxFG repeats can displace importin-beta or its S. cerevisiae homologue, Kap95, bound to GLFG repeats. Addition of soluble GLFG repeats decreases the rate of nuclear protein import in digitonin-permeabilized HeLa cells, indicating that this interaction has a role in the translocation of carrier-cargo complexes through nuclear pores. The binding of GLFG and FxFG repeats to overlapping sites on importin-beta indicates that functional differences between different repeats probably arise from differences in their spatial organization.

Active Transport, Cell Nucleus↗
PubMed2002

Evidence for a posttranscriptional role of a TFIIICalpha-like protein in Chironomus tentans.

We have cloned and sequenced a cDNA that encodes for a nuclear protein of 238 kDa in the dipteran Chironomus tentans. This protein, that we call p2D10, is structurally similar to the alpha subunit of the general transcription factor TFIIIC. Using immunoelectron microscopy we have shown that a fraction of p2D10 is located at sites of transcription, which is consistent with a possible role of this protein in transcription initiation. We have also found that a large fraction of p2D10 is located in the nucleoplasm and in the nuclear pore complexes. Using gel filtration chromatography and coimmunoprecipitation methods, we have identified and characterized two p2D10-containing complexes that differ in molecular mass and composition. The heavy p2D10-containing complex contains at least one other component of the TFIIIC complex, TFIIIC-epsilon. Based on its molecular mass and composition, the heavy p2D10-containing complex may be the Pol III holoenzyme. The light p2D10-containing complex contains RNA together with at least two proteins that are thought to be involved in mRNA trafficking, RAE1 and hrp65. The observations reported here suggest that this new TFIIIC-alpha-like protein is involved in posttranscriptional steps of premRNA metabolism in Chironomus tentans.

Amino Acid Motifs↗
PubMed2002

Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner.

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.

Animals↗
PubMed2000

Calcium, ATP and nuclear pore channel gating.

Nuclear envelope (NE) cisternal Ca2+ and cytosolic ATP are required for nuclear-pore-complex-(NPC-) mediated transport of DNAs, RNAs, transcription factors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. The large ion conductance (gamma) activity of the NPC channel (NPCC) is blocked by the NPC monoclonal antibody mAb414, known to block MMT, and is also silenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. To test the role of Ca2+ and ATP in NPCC activity, we carried out the present patch-clamp study with the pipette attached to the outer NE membrane of nuclei isolated from cultured Dunning G prostate cancer cells. Our investigations demonstrate that in these isolated nuclei neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. However, when simultaneously applied to the bath and pipette, they transiently silence NPCC activity through stimulation of MMT by raising the Ca2+ concentration in the NE cisterna ([Ca2+]NE). Our fluorescence microscopy observations with nuclear-targeted macromolecular fluorochromes (B-phycoerythrin and plasmid for the enhanced green fluorescence protein EGFP, pEGFP-C1) and with FITC-labeled RNA support the view that channel silence accompanies MMT. Repeated Ca2+ loading of the NE with Ca2+ and ATP, after unloading with 1-5 microM inositol 1,4,5-trisphosphate (IP3), thapsigargin (TSG) or 5 mM BAPTA or EGTA, failed to affect channel gating. This result indicates that other factors are involved in this phenomenon and that they are exhausted during the first cycle of NE Ca2+ loading/unloading--in agreement with current theories of NPC-mediated MMT. The results explain how Ca2+ and IP3 waves may convert the NE into an effective Ca2+ barrier and, consequently, affect the regulation of gene activity and expression through their feedback on MMT and NPCC gating. Thus, [Ca2+]NE regulation by intracellular messengers is an effective mechanism for synchronizing gene activity and expression to the cellular rhythm.

Adenosine Triphosphate↗
PubMed1997

Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.

NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.

Animals↗
PubMed2002

The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive.

After being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22 degrees C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

Active Transport, Cell Nucleus↗
PubMed1999

Structural view of the Ran-Importin beta interaction at 2.3 A resolution.

Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.

Amino Acid Sequence↗
PubMed1982

Dynamic aspects of the supramolecular organization of intermediate filament networks in cultured epidermal cells.

We have shown, by indirect immunofluorescence microscopy using an antiserum against the mouse keratin subunit K2 and by electron microscopy, that transformed (PAM) and primary (PME) mouse epidermal cells possess extensive networks of IF bundles. Following trypsinization and replating of PAM cells, IF bundles are seen to move as a continuous network from a perinuclear zone into the peripheral cytoplasmic regions. In PAM cells lysed in high-ionic-strength solutions containing Triton X-100 and DNAase-1, IF bundles appear to be closely associated with nuclear envelope remnants and, in some cases, appear to be attached to nuclear pore complexes. PME cells cultivated in low Ca2+-containing medium possess perinuclear birefringent arrays of IF bundles. Within 2 hours of switching the cells to normal Ca2+ levels, the PME IF bundle network moves towards and establishes contact with the cell surface as desmosomes form. Live cells observed by phase contrast and fixed cells observed by immunofluorescence microscopy demonstrate that desmosomes can be distinguished as dark bands separating neighboring cells. There is little difference between the major proteins seen in SDS-polyacrylamide gel profiles of isolated IF bundle networks from PME cells before and after the Ca2+ switch. Therefore, a reorganization of relatively insoluble membrane-associated protein following the Ca2+ switch may be involved in desmosome formation. The isolated IF networks from PAM cells differ in protein composition compared to the PME IF networks. This may be related to the greatly reduced number of desmosomes in PAM cells. The IF bundle system in epidermal cells appears to be involved in shape formation, shape maintenance, the establishment of desmosomes, nuclear centration, and cell-cell contact.

Animals↗
PubMed2004

The evolutionarily conserved Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with REF protein and acts as an RNA export factor.

ORF57 (MTA) one of the earliest Kaposi's sarcoma-associated herpesvirus (KSHV) regulatory proteins to be expressed is essential for virus lytic replication. A counterpart is present in every herpesvirus sequenced, indicating the importance of this signature viral protein and those examined act post-transcriptionally, affecting RNA splicing and transport. In KSHV-infected cells, ORF57 protein was present in a complex with REF (Aly) and TAP (NXF1), factors involved in cellular mRNA export. The ORF57 N-terminal region interacts with REF, whereas both N- and C-terminal domains of REF interact with ORF57. The ORF57-REF interaction was direct, whereas TAP appeared to be recruited via REF. In somatic cells, ectopically expressed ORF57 protein was shown to function as a CRM1-independent nuclear mRNA export factor, promoting export of mRNAs that are poor substrates for splicing. The gamma-herpesvirus ORF57 protein, and its alpha-1 herpesvirus ICP27 counterpart both export RNA through pathways involving REF and TAP proteins, although divergence of these herpesvirus subfamilies occurred some 180-210 million years ago. The TAP-mediated cellular mRNA export pathway is CRM1-independent. However, human immunodeficiency virus type 1 Rev protein-mediated RNA export, which is CRM1-dependent, was considerably inhibited by ORF57, suggesting that Rev and ORF57 compete for a common export component. These data strengthen arguments that TAP and CRM1 pathways converge in accessing similar components of the nuclear pore complex. We propose that ORF57-mediated RNA export may use different export factors to accommodate the KSHV-infected host cell environments, for example, in B-cells or endothelial cells and during the different phases of lytic virus replication.

Active Transport, Cell Nucleus↗
PubMed1996

Regulation of mRNA export in response to stress in Saccharomyces cerevisiae.

The response of eukaryotic cells to heat shock and other forms of stress occurs at both transcriptional and post-transcriptional levels. We used in situ hybridization to determine whether stress affected the subcellular distribution of poly(A)+ RNA in Saccharomyces cerevisiae. Following induction of stress by either heat shock (42 degrees C) or addition of a high concentration of ethanol (10%), the nucleocytoplasmic export of most poly(A)+ RNA was blocked. In situ hybridization indicated that heat-inducible SSA4 and SSA1 mRNAs were exported from nuclei under these same conditions. On the other hand, both GAL1 and URA3 transcripts expressed from the SSA4 promoter accumulated in nuclei following heat shock. Sequences within either the 5' 1600 or the 3' 500 nucleotides of SSA4 mRNA were sufficient to direct GAL1 mRNA to the cytoplasm during stress. The export of SSA4 mRNA following stress required functional nuclear pore complexes, as SSA4 mRNA accumulated in nuclei following heat shock of cells containing temperature-sensitive nucleoporins. However, the selective export of SSA4 mRNA was maintained in heat-shocked cells carrying temperature-sensitive alleles of RNA1, PRP20, or an inducible dominant-negative allele of GSP1, the S. cerevisiae homolog of RAN/TC4. The results reported here suggest that there is selective export of mRNA in yeast.

Adenosine Triphosphatases↗
PubMed1990

Visualization of transport-related configurations of the nuclear pore transporter.

The transport of macromolecules between the cytoplasm and nucleus of the cell is mediated by the nuclear pore complex (NPC). In this study, details of the central transporter assembly within NPCs have been examined by cryoelectron microscopy, image processing, and classification analysis. The NPC transporter in isolated amphibian nuclei appears to adopt a minimum of four transport-related configurations including: (a) a putative closed form with a 90-100 A diameter central pore, (b) a docked form with material aligned over the pore, (c) an open form with substrates apparently caught "in transit," and (d) an open form with an enlarged pore. This data confirms previous observations on NPC transporters labeled with nucleoplasmin-gold (Akey, C.W., and D.S. Goldfarb. 1989. J. Cell Biol. 109:971-982) and allows a working model of the central NPC transporter to be proposed. The model is comprised of two supramolecular irislike assemblies which open asynchronously to provide an expanded pore for translocation while maintaining transport fidelity.

Animals↗
PubMed2001

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

Animals↗
PubMed2005

Glucocorticoids remodel nuclear envelope structure and permeability.

The present study describes glucocorticoid induced remodelling of nuclear envelope (NE) structure and permeability. A glucocorticoid analogue, triamcinolone acetonide (TA), is injected into Xenopus laevis oocytes that express an exogeneous glucocorticoid receptor (GR). Electrical, fluorescence and nano-imaging techniques are applied to study the permeability and the structure of the NE at 5 and 60 minutes after injection of TA. A remarkable dilation of nuclear pore complexes (NPCs), a rearrangement of NPC distribution and a significant increase of NE permeability for ions and fluorescent 20 kDa dextran are observed within 5 minutes of TA exposure. At regular distances on local NE patches, NPCs seem to adjoin forming clusters each consisting of several hundred NPCs. Interestingly, at the same time of exposure, hydrophobicity of NPC central channels and NPC-free NE surface increases. The changes in permeability and structure are transient as the NE permeability returns to its initial state within 60 minutes. In conclusion, the NE is a barrier of high plasticity sensitive to hydrophobic molecules. Remodelling of NE structure and permeability is a prerequisite for mediating physiological actions of glucocorticoids.

Animals↗
PubMed2005

Pushing the envelope: structure, function, and dynamics of the nuclear periphery.

The nuclear envelope (NE) is a highly specialized membrane that delineates the eukaryotic cell nucleus. It is composed of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) and, in metazoa, the lamina. The NE not only regulates the trafficking of macromolecules between nucleoplasm and cytosol but also provides anchoring sites for chromatin and the cytoskeleton. Through these interactions, the NE helps position the nucleus within the cell and chromosomes within the nucleus, thereby regulating the expression of certain genes. The NE is not static, rather it is continuously remodeled during cell division. The most dramatic example of NE reorganization occurs during mitosis in metazoa when the NE undergoes a complete cycle of disassembly and reformation. Despite the importance of the NE for eukaryotic cell life, relatively little is known about its biogenesis or many of its functions. We thus are far from understanding the molecular etiology of a diverse group of NE-associated diseases.

Animals↗