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PubMed2004

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans.

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.

3T3 Cells↗
PubMed2003

Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore.

The nuclear poly(A)-binding protein, PABPN1, has been previously shown to regulate mRNA poly(A) tail length and to interact with selected proteins involved in mRNA synthesis and trafficking. To further understand the role of PABPN1 in mRNA metabolism, we used cryo-immunoelectron microscopy to determine the fate of PABPN1 at various stages in the assembly and transport of the Chironomus tentans salivary gland Balbiani ring (BR) mRNA ribonucleoprotein (mRNP) complex. PABPN1 is found on BR mRNPs within the nucleoplasm as well as on mRNPs docked at the nuclear pore. Very little PABPN1 is detected on the cytoplasmic side of the nuclear envelope, suggesting that PABPN1 is displaced from mRNPs during or shortly after passage through the nuclear pore. Surprisingly, we also find PABPN1 associated with RNA polymerase II along the chromatin axis of the BR gene. Our results suggest that PABPN1 binds to the polymerase before, at, or shortly after the start of transcription, and that the assembly of PABPN1 onto the poly(A) tail may be coupled to transcription. Furthermore, PABPN1 remains associated with the released BR mRNP until the mRNP is translocated from the nucleus to the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed1981

Temperature-induced changes in nuclear pore complex frequencies, nuclear envelope surface areas, and nuclear volumes in light-synchronized Euglena.

An autotrophic culture of Euglena, synchronized using a day:night (D:N), 14:10-h cycle, was subjected to a 21.5 leads to 31.5 degrees C temperature shift and then to a reversed shift in temperature after three D:N cycles at 31.5 degrees C. Nuclear pore complex (NPC) number per square micrometre and nuclear surface area and volume determinations were made on G1 cells at various intervals. Cells sampled immediately prior to the 21.5 leads to 31.5 degrees C shift had a mean value of 37.68 NPC . micron-2 nuclear envelope surface area, 30.40 NPCs/micron2 after three D:N cycles at 31.5 degrees C and 39.98 NPCs/micron2 after three D:N cycles at the resumed culture temperature of 21.5 degrees. Thus temperature changes affect NPC numbers per square micrometre and these changes are reversible. Mean nuclear surface area was 125.76 micron2 immediately prior to the 21.5 leads to 31.5 degrees C shift, and decreased over two D:N cycles at 31.5 degrees C to 101.30 micron2 by the end of the third D:N cycle. Nuclear envelope surface area, one and two D:N cycles after the 31.5 leads to 21.5 degrees C shift, was approximately equal that prior to the 21.5 leads to 31.5 degrees C shift. After the third D:N cycle, however, nuclear surface area had increased to 173.05 micron2. The changes in nuclear surface area resulted in large differences in the estimates of the total number of NPCs per nucleus. Euglena immediately prior to the 21.5 leads to 31.5 degrees C temperature shift had 4739 NPCs/nucleus; immediately prior to the 31.5 leads to 21.5 degrees C shift had 3079 NPCs/nucleus; and had 6919 NPCs/nucleus at 21.5 degrees C and three D:N cycles after the 31.5 leads to 21.5 degrees C shift. Estimates of the number of NPCs per cubic micrometre of nuclear volume were almost identical between these samples.

Animals↗
PubMed1998

Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export.

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

Animals↗
PubMed1994

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.

Amino Acid Sequence↗
PubMed2002

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes.

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.

Binding Sites↗
PubMed2004

Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex.

Nuclear pore complexes (NPCs) are supramolecular protein pores that traverse the nuclear envelope and form the only known direct route of transport between the cytoplasmic and nuclear spaces. Detailed studies have identified both active and passive mechanisms of transport through the NPC and structural studies have revealed its three-dimensional architecture. Under certain conditions, structural studies have found evidence for a mass in the central pore of the NPC whose identity remains unclear. Some studies suggest this mass represents cargo caught in transit, while others suggest it is an integral component of the NPC, the position of which is sensitive to sample conditions. Regardless of its identity, previous studies have shown that the central mass location within the NPC pore is influenced by the presence of calcium in the cisternal spaces of the nuclear membrane. Specific depletion of these calcium stores through inositol 1,4,5-trisphosphate (IP(3)) receptor activation leads to the apparent displacement of the central mass towards both the cytoplasmic and nucleoplasmic sides of the NPC. Whether the central mass is cargo or a NPC component, these observations may offer interesting insights linking transport and calcium signaling pathways. Here, we show that ryanodine (Ry) receptors are also present in the nuclear envelope of Xenopus laevis oocytes, and their specific activation can affect the conformational state of the NPC. Although previously undetected, Western blot analysis of isolated oocyte nuclei reveals the presence of Ry receptors in the nuclear envelope, albeit in low abundance. Extensive atomic force microscopy (AFM) studies at the single pore level of isolated, fixed nuclei reveal changes in the NPC conformational state following treatments that stimulate Ry receptor activity. At resting calcium levels ( approximately 200 nM Ca(2+)), the central mass within the lumen of the NPC is recessed 5.3 nm below the cytoplasmic rim of the NPC. Following treatment with 10 nM ryanodine, the central mass displaces towards the cytoplasmic face occupying a new position only 2.9 nm below the cytoplasmic rim. Interestingly, at high ryanodine concentrations (20 microM), which are reported to deactivate Ry receptors, the central mass is observed to return to the recessed position, 5.4 nm below the cytoplasmic rim. Treatments with caffeine also lead to large changes in the NPC conformation, confirming the link to specific activation of Ry receptors. These observations are consistent with a new mechanism of NPC regulation in which specific activation of Ry receptors located in the nuclear envelope can modulate cisternal calcium levels, leading to changes in the NPC conformation. Together with previous studies, it now appears that both IP(3) and Ry receptors are present in the nuclear envelope of Xenopus oocytes and are capable, through activation, of indirectly influencing the conformational state of the NPC.

Animals↗
PubMed2005

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex.

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.

Animals↗
PubMed2006

From the trap to the basket: getting to the bottom of the nuclear pore complex.

Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the double-membraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of high-resolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed 'minimalist' NPC.

Cytoplasm↗
PubMed2005

The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation.

The metazoan nuclear envelope (NE) breaks down and reforms at each mitosis. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the reforming NE at the end of mitosis. Using in vitro NE assembly assays, we show that one of the two transmembrane nucleoporins, pom121, is essential for NE formation, whereas the second, gp210, is dispensable. Depletion of either pom121-containing membrane vesicles or the protein alone does not affect vesicle binding to chromatin but prevents their fusion to form a closed NE. When the Nup107-160 complex, which is essential for integration of NPCs into the NE, is also depleted, pom121 becomes dispensable for NE formation, suggesting a close functional link between NPC and NE formation and the existence of a checkpoint that monitors NPC assembly state.

Animals↗
PubMed2026

ELYS associates with distinct DNA sequence environments during post-mitotic nuclear pore reassembly.

Nuclear pore complexes (NPCs) contribute to genome organization and cell identity, yet how post-mitotic NPC assembly is coordinated with chromatin architecture remains unclear. Here, we show that the nucleoporin ELYS preferentially associates with chromatin regions displaying distinct intrinsic DNA sequence features that are not explained by the repressive histone marks examined here. ELYS-bound regions are enriched for AT-rich sequences, whereas ELYS binding at super-enhancer-associated loci shift toward GC-rich sequence composition, revealing distinct sequence environments. These findings indicate that ELYS localization is associated with distinct intrinsic DNA sequence features and suggest a mechanism by which nuclear pore-associated architecture restores transcriptional programs after mitosis.

Journal Article↗
PubMed2004

Imaging of single-molecule translocation through nuclear pore complexes.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.

Antigens, Viral, Tumor↗
PubMed1998

The nuclear pore complex.

The nuclear pore complex is the largest supramolecular complex that assembles in the eukaryotic cell. This structure is highly dynamic and must disassemble prior to mitosis and reassemble after the event. The directed movement of macromolecules into and out of the nucleus occurs through the nuclear pore complex, a potentially regulatory point for translocation. Using biochemical and genetic approaches, several nuclear pore complex proteins from yeast and vertebrates have been well characterized. Although very little is known about plant nuclear pore proteins, research is providing new information that indicates that plant nuclear pore complexes may have some unique features.

Amino Acid Sequence↗
PubMed1978

Change of the nuclear pore frequency during the nuclear cycle of Physarum polycephalum.

The change of the nuclear pore frequency during the nuclear cycle of synchronous plasmodia of Physaru- polycephalum was investigated. Counts were made on platinum-carbon replicas of isolated nuclei. Pore numbers varied markedly at any given time of the nuclear cycle, possibly due to the variable DNA contents of the nuclei. The average pore frequency per nucleus increased from 336 at 1 h after mitosis to 770 at 50 min before the subsequent mitosis. The results suggest a quantitative relation between the nuclear DNA content and pore frequency. This is compatible with the hypothesis that pore complexes serve as attachment sites for nuclear DNA.

Microscopy, Electron↗
PubMed1997

Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.

Animals↗
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↗
PubMed1993

Isolation of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.

Cell Fractionation↗
PubMedDate not supplied

Calcium regulation of nuclear pore permeability.

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.

Animals↗