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PubMed1996

Genetic analysis of macromolecular transport across the nuclear envelope.

Numerous factors that promote movement of macromolecules in and out of the nucleus have now been identified. These include both soluble cytoplasmic and nucleoplasmic proteins and proteins of the nuclear pore complex (NPC). Genetic analyses of the nuclear transport process in the model organism, the budding yeast Saccharomyces cerevisiae, have revealed remarkable conservation of all of these factors. In addition, important clues as to how these factors promote the unique bidirectional movement across the NPC have emerged from studies of yeast. We summarize the characterization and genetic interactions of the soluble transport factors and present data to illustrate how genetic experiments can be used to further define the import and export pathways.

Biological Transport, Active↗
PubMed1989

Nuclear granules recognized by some monoclonal antibodies against intermediate filament protein locate on chromosomes during mitosis.

AC54 monoclonal antibody (MAb), an anti-desmin MAb, recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells. To investigate nuclear granules, similar MAbs were obtained by using desmin fraction as an antigen. Among them, DSB389 MAb recognized mainly nuclear granules in HeLa and rat liver cells. The nuclear granules in HeLa cells were aligned in arrays, sometimes connected by, or part of, a rope-like structure, and stable against treatment with 0.5% Triton X-100 and 2 M NaCl. They located on or around the chromosomes during mitosis. Essentially the same results were obtained with DSB860 and AC54 MAbs. The distribution of the granules in liver nuclei recognized by DSB389 MAb was similar to that of DNA and was different from that of the nuclear pore complexes. The biological significance of the nuclear granules is discussed.

Animals↗
PubMed1998

Mutations in fission yeast Cut15, an importin alpha homolog, lead to mitotic progression without chromosome condensation.

Chromosome condensation is a major mitotic event. Fission yeast mutations in topoisomerase II and condensin subunits produce the characteristic 'cut' phenotypes, in which the septum bisects the nuclear material in the absence of normal condensation and sister chromatid separation. We show here that the same condensation defect is produced in cut15 temperature-sensitive mutants at the restrictive temperature (36 degrees C). The gene product of cut15+ is, surprisingly, very similar to importin alpha, which binds proteins containing a nuclear localization signal (NLS) and forms the heterodimer with importin beta that mediates translocation through the nuclear pore complex. We show that in a nuclear import assay, purified Cut15 protein behaved identically to mammalian importin alpha but mutant Cut15 did not. Mutant Cut15 failed to bind an NLS-containing protein in vitro but could still bind importin beta. Unexpectedly, however, NLS proteins were imported into the nucleus in cut15 mutants. Cut15 is thus essential for mitotic chromosome condensation, but its role in nuclear import might be dispensable. Green fluorescent protein (GFP)-tagged Cut15 was enriched within the nucleus specifically during prometaphase-metaphase, so the interaction of Cut15 with nuclear NLS proteins during mitosis might be important for condensation.

Animals↗
PubMed1989

Identification of specific binding proteins for a nuclear location sequence.

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.

Amino Acid Sequence↗
PubMed1999

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.

Allosteric Regulation↗
PubMed1992

The nuclear membrane.

The nuclear membrane forms a major barrier within the cell, permitting levels of regulation not found in prokaryotes. The dynamics and diverse functions of the nuclear membrane and its associated structures are considered in this review. The role of the nuclear pore complex in selective transport across the nuclear membrane has been studied to a considerable degree; however, many crucial questions remain. Components of a signal transduction mechanism are associated with the nucleus, suggesting that nuclear functions may be influenced directly by this system. The involvement of the heat shock cognate protein Hsc70 in nuclear protein import is discussed, and a specific signal-presentation role for this protein is proposed.

Animals↗
PubMedDate not supplied

Morphological changes and nuclear pore clustering during nuclear degradation in differentiating bovine lens fibre cells.

The programmed degradation of organelles is a characteristic feature of lens fibre cell differentiation. Due to the large number of similarities between the programmed organelle loss during lens development and the changes to organelles in apoptosis, lens cell differentiation has been suggested to share a common basis with programmed cell death. This study was aimed at characterising the morphological changes to the nucleus during cellular differentiation in the bovine lens at the ultrastructural level. Progressive shrinkage of the nucleus is accompanied by clumping and marginalisation of the chromatin to the nuclear periphery. Additionally, the fate of another key component of the nuclear envelope--the nuclear pore complexes--was followed. In parallel to the shrinkage of the nucleus, the nuclear pores progressively cluster into large aggregates that associate with the condensed DNA. These observations in differentiating lens fibres mirror the situation in cells undergoing apoptosis and thus provide additional data supporting a common basis between the two processes.

Animals↗
PubMed1977

Nuclear envelope transport capacity and the cell cycle in yeast (Saccharomyces cerevisiae).

Changes in the nuclear envelope transport capacity, as measured by the number of nuclear pore complexes/unit nuclear volume/cell, were followed during the Saccharomyces cerevisiae cell cycle using data obtained by freeze-fracture electron microscopy. Pore number per unit nuclear volume decreased sharply in early G0, remained steady from mid-GO through S to G2, and showed a further slight decrease at M and G1. These periods of decline apparently resulted from nuclear enlargement without sufficient formation of new nuclear pore complexes to maintain the pore number to nuclear volume ratio. However, marked nuclear pore formation did accompany both increases in nuclear volume. The significance of these changes in relation to other events in the cell cycle is discussed. The validity of using nuclear pore number/unit nuclear volume and other pore number data as indices of nuclear envelope transport capacity and cell activity is critically examined.

Biological Transport↗
PubMed1996

Epidermal ultrastructure of the southern right whale calf (Eubalaena australis).

An ultrastructural analysis by transmission and scanning electron microscopy was carried out on normal epidermis of six southern right whale (Eubalaena australis) calves which stranded over a period of several months at Peninsula Valdes, Argentina. This was undertaken to 1) provide the first normal skin ultrastructural data on this highly endangered species which is known to display skin pathology in some instances, and 2) to elucidate further the integumentary specializations which have developed in diving marine mammals. Southern right whale lipokeratinocytes demonstrated parakeratosis and numerous intracellular lipid bodies, keratin and melanosomes, as reported for other cetacean species, but showed several unique ultrastructural features as well. These included a high prevalence of intranuclear inclusion bodies resembling small fragments of cytoplasmic keratin, and close structural relationship between cytoplasmic lipid droplets and the nucleus. The subcellular morphology supported the concept of possible nuclear import of cytoplasmic keratin and lipid metabolites through enlargements of the nuclear pore complex or other disruptions of the nuclear envelope. The light microscopy and scanning electron microscopy also revealed an irregular contour of the lipokeratinocytes which comprised the thick stratum externum, and surface flaking of the outermost cells which were covered by stubby microvillous-like remnants of intercellular junctions. These results thus suggest that the long-tem aquatic evolution of this cetacean species has resulted in a number of integumentary specializations and that investigation of their submicroscopic cytology may help elucidate the general cell biology of nuclear-cytoplasmic interactions.

Animals↗
PubMed2002

The SUMO E3 ligase RanBP2 promotes modification of the HDAC4 deacetylase.

Transcriptional repression mediated through histone deacetylation is a critical component of eukaryotic gene regulation. Here we demonstrate that the class II histone deacetylase HDAC4 is covalently modified by the ubiquitin-related SUMO-1 modifier. A sumoylation-deficient point mutant (HDAC4-K559R) shows a slightly impaired ability to repress transcription as well as reduced histone deacetylase activity. The ability of HDAC4 to self-aggregate is a prerequisite for proper sumoylation in vivo. Calcium/calmodulin-dependent protein kinase (CaMK) signalling, which induces nuclear export, abrogates SUMO-1 modification of HDAC4. Moreover, the modification depends on the presence of an intact nuclear localization signal and is catalysed by the nuclear pore complex (NPC) RanBP2 protein, a factor newly identified as a SUMO E3 ligase. These findings suggest that sumoylation of HDAC4 takes place at the NPC and is coupled to its nuclear import. Finally, modification experiments indicate that the MEF2-interacting transcription repressor (MITR) as well as HDAC1 and -6 are similarly SUMO modified, indicating that sumoylation may be an important regulatory mechanism for the control of transcriptional repression mediated by both class I and II HDACs.

Animals↗
PubMed2005

The chromatoid body in spermatogenesis.

All germ cells throughout the animal kingdom contain cytoplasmic cloud-like accumulations of material called nuage. Polar bodies in Drosophila oocytes are probably the best known forms of nuage. In spermatogenic cells, the nuage is called chromatoid body (CB). In early spermatids of the rat, it has a diameter of 1-1.5 microm and a finely filamentous lobular structure. Typically, it is associated with a multitude of vesicles. It is first clearly seen in mid- and late pachytene spermatocytes as an intermitochondrial dense material. During early spermiogenesis it is seen near the Golgi complex and frequently connected by material continuities through nuclear pore complexes with intranuclear particles. In living cells, the CB moves around the Golgi complex and has frequent contacts with it. The CB also moves perpendicularly to the nuclear envelope, and even through cytoplasmic bridges to the neighbour spermatids. One of the major components of the CB is a DEAD-box RNA helicase VASA that belongs to a class of proteins thought to act as RNA chaperones. It is a general marker of all germ cells and best characterized in Drosophila. The mouse VASA homologue was recently used as a marker of sperm formation from embryonic stem cells. It becomes generally accepted that the CB with its associated structures constitute a mechanism of post-transcriptional processing and storage of several mRNA species that are shared between neighbour cells and used for translation when the genome of the spermatids becomes inactive.

Animals↗
PubMed2003

Importin beta-depending nuclear import pathways: role of the adapter proteins in the docking and releasing steps.

Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin beta. However, due to the presence of different import signals, the adapter protein of the imported molecules and importin beta is different for each pathway. Although the adapter for cNLS-protein is importin alpha, the adapter for U1 snRNP is snurportin1 (SPN1). Herein, we show that the use of distinct adapters by importin beta results in differences at the docking and releasing step for these two import pathways. Nuclear pore complex (NPC) docking of U1 snRNP but not of cNLS-protein was inhibited by an anti-CAN/Nup214 antibody. Thus, the initial NPC-binding site is different for each pathway. Pull-down assays between immobilized SPN1 and two truncated forms of importin beta documented that SPN1 and importin alpha have different binding sites on importin beta. Importin beta fragment 1-618, which binds to SPN1 but not to importin alpha, was able to support the nuclear import of U1 snRNPs. After the translocation through the NPC, both import complexes associated with the nuclear side of the NPC. However, we found that the nature of the importin beta-binding domain of the adapters influences the release of the cargo into the nucleoplasm.

Active Transport, Cell Nucleus↗
PubMed2004

The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo.

RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.

DNA Primers↗
PubMed2006

Composition of the plant nuclear envelope: theme and variations.

The nuclear envelope is the hallmark of all eukaryotic cells, separating the nucleoplasm from the cytoplasm. At the same time, the nuclear envelope allows for the controlled exchange of macromolecules between the two compartments through nuclear pores and presents a surface for anchoring and organizing cytoskeletal components and chromatin. Although our molecular understanding of the nuclear envelope in higher plants is only just beginning, fundamental differences from the animal nuclear envelope have already been found. This review provides an updated investigation of these differences with respect to nuclear pore complexes, targeting of Ran signalling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis.

Active Transport, Cell Nucleus↗
PubMed1999

A Crm1p-independent nuclear export path for the mRNA-associated protein, Npl3p/Mtr13p.

mRNA export involves association of mRNAs with nucleoplasmic proteins, delivery to the nuclear pore complex, translocation to the cytoplasm, and reimport of recycling components. Many yeast mutants inhibit mRNA export, but there is little information concerning the RNA carriers and steps of transport that they affect. The hnRNP/serine-arginine-rich-like protein, Npl3p/Mtr13p, binds poly(A)+ RNA and shuttles between the nucleus and cytoplasm. Its export accelerates on inhibition of RNA synthesis. In vivo tests show that its export requires two proteins with putative leucine-rich nuclear export signals: Gle1p, Mex67p, and several additional nuclear and nuclear pore complex-associated proteins. Surprisingly, a nonnuclear pool of an import factor (the importin alpha homologue, Srp1p) is also required. Changes in the methylation status of Npl3p do not correlate with its nucleocytoplasmic distribution. A crm1 mutant that inhibits export of proteins with leucine-rich nuclear export signals and mRNAs does not inhibit Npl3p export. Moreover, several proteins needed for Npl3p export are not needed for export of a typical Crm1p cargo. Thus, Npl3p export requires only a subset of proteins implicated in mRNA export, suggesting that more than one mRNA export path exists. A distinct group of mutants, including a mutation of a member of the importin beta superfamily, inhibits Npl3p reimport from the cytoplasm.

Biological Transport↗
PubMed2004

Distinct functional domains within nucleoporins Nup153 and Nup98 mediate transcription-dependent mobility.

Despite the apparent overall structural stability of the nuclear pore complex during interphase, at least two nucleoporins have been shown to move dynamically on and off the pore. It is not yet certain what contribution nucleoporin mobility makes to the process of nuclear transport or how such mobility is regulated. Previously, we showed that Nup98 dynamically interacts with the NPC as well as bodies within the nucleus in a transcription-dependent manner. We have extended our studies of dynamics to include Nup153, another mobile nucleoporin implicated in RNA export. In both cases, we found that although only one domain is essential for NPC localization, other regions of the protein significantly affect the stability of association with the pore. Interestingly, like Nup98, the exchange of Nup153 on and off the pore is inhibited when transcription by Pol I and Pol II is blocked. We have mapped the regions required to link Nup98 and Nup153 mobility to transcription and found that the requirements differ depending on which polymerases are inhibited. Our data support a model whereby transcription of RNA is coupled to nucleoporin mobility, perhaps ultimately linking transport of RNAs to a cycle of remodeling at the nuclear pore basket.

Active Transport, Cell Nucleus↗
PubMed2006

Cellular localization of 17 natural mutant variants of ALADIN protein in triple A syndrome - shedding light on an unexpected splice mutation.

The triple A syndrome is a complex and multisystemic autosomal recessive disease with the 3 main symptoms of adrenal insufficiency, alacrima, and achalasia accompanied by neurological impairment. Mutations in the AAAS gene on chromosome 12q13 are responsible for the disorder. AAAS encodes a protein named ALADIN, which belongs to the family of WD-repeat-containing proteins and has been shown to localize to nuclear pore complexes. The function of the protein is not clear. It is supposed that ALADIN plays an important role in RNA and (or) protein trafficking between the nucleus and cytoplasm. With transfection experiments, we analyzed the cellular localization of the wild-type and 17 natural mutant variants (9 missense, 5 nonsense, 3 frameshift mutations) of ALADIN. We show that most mutations cause mislocalization of the mutant ALADIN proteins in the cytoplasm. In contrast, some variants with mutations located at the N-terminus (Q15K, L25P) and 3 artificial C-terminus mutations (Q490X, R493X, and V497X) remain at the nuclear pore. Using a patient cell line, we show that the mutation 43C>A in exon 1 does not cause a missense mutation Q15K but, rather, results in aberrant splicing.

Addison Disease↗
PubMed2004

Visualization of a highly organized intranuclear network of filaments in living mammalian cells.

For 30 years, the mammalian cell nucleus has been hypothesized to contain a filamentous framework, the nuclear matrix or karyoskeleton, which regulates nuclear structure and function. However, such an organized network of filaments has never been observed in living cells. Here we show that human Cdc14B phosphatase in living cells tightly associates with long filaments that begin at the nucleolar periphery and extend to the nuclear envelope, frequently making close connections with nuclear pore complexes. We demonstrate that Cdc14B contains a bipartite signal that directs it to the intranuclear filaments, and we also detect a small amount of Cdc14B on interphase and mitotic centrosomes. Furthermore, we show that Cdc14B is critical for the maintenance of proper nuclear structure together with polo-like kinase Plk1. This work provides the first direct evidence for the existence of an intranuclear filamentous framework in living mammalian cells and implicates Cdc14B in the control of mammalian nuclear architecture.

Animals↗