Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy

1995 ◽  
Vol 108 (9) ◽  
pp. 2963-2972 ◽  
Author(s):  
M. Grote ◽  
U. Kubitscheck ◽  
R. Reichelt ◽  
R. Peters
Keyword(s):  
Cross Sections ◽  
Nuclear Pore Complex ◽  
Structural Model ◽  
Human Keratinocytes ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Pore Protein ◽  
Ultrathin Sections ◽  
Lowicryl K4m ◽  
Secondary Antibodies

Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominatly to the controversely discussed ‘central granule’. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.

scholarly journals Comparative Spatial Localization of Protein-A-Tagged and Authentic Yeast Nuclear Pore Complex Proteins by Immunogold Electron Microscopy

2000 ◽  
Vol 129 (2-3) ◽  
pp. 295-305 ◽  
Author(s):  
Birthe Fahrenkrog ◽  
John P. Aris ◽  
Eduard C. Hurt ◽  
Nelly Panté ◽  
Ueli Aebi
Keyword(s):  
Electron Microscopy ◽  
Nuclear Pore Complex ◽  
Protein A ◽  
Spatial Localization ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore

scholarly journals Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins.

1989 ◽  
Vol 108 (6) ◽  
pp. 2059-2067 ◽  
Author(s):  
J P Aris ◽  
G Blobel
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Nuclear Pore Complex ◽  
Staining Pattern ◽  
Subcellular Fractions ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Fraction ◽  
Pore Complexes

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.

scholarly journals Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex.

1994 ◽  
Vol 127 (6) ◽  
pp. 1515-1526 ◽  
Author(s):  
D A Byrd ◽  
D J Sweet ◽  
N Panté ◽  
K N Konstantinov ◽  
T Guan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Liver ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Coiled Coil ◽  
In Vitro Translation ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Cytoplasmic Surface

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, which reacts with novel nuclear pore complex (NPC) antigens that are not O-glycosylated. By immunofluorescence staining of cultured cells, RL30 reacts exclusively with the NE in a punctate pattern that largely coincides with that of identified NPC proteins. RL30 labels only the cytoplasmic surface of the NPC in immunogold electron microscopy, predominantly in peripheral regions nearby the cytoplasmic ring. In immunoblots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NEs that are likely to be proteolytic products of p265. Sequencing of peptides from the 175- and 265-kD RL30 antigens of rat liver revealed that they are both closely related to human Tpr, a protein whose amino-terminal 150-250 amino acids appear in oncogenic fusions with the kinase domains of the met, trk, and raf protooncogenes. We found that in vitro translation of human Tpr mRNA yields a major 265-kD band. Considered together, these data indicate that the 265-kD RL30 antigen in the NPC is the rat homologue of Tpr. Interestingly, Tpr contains an exceptionally long predicted coiled coil domain (approximately 1600 amino acids). The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC reassembly at the end of mitosis, Tpr becomes concentrated at the NE significantly later than O-linked glycoproteins, including p62. This indicates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.

scholarly journals Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex

2017 ◽  
Vol 74 (11) ◽  
pp. 2107-2125 ◽  
Author(s):  
Flavie Courjol ◽  
Thomas Mouveaux ◽  
Kevin Lesage ◽  
Jean-Michel Saliou ◽  
Elisabeth Werkmeister ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Nuclear Pore Protein ◽  
Pore Protein

scholarly journals Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins.

1996 ◽  
Vol 134 (3) ◽  
pp. 589-601 ◽  
Author(s):  
T Hu ◽  
T Guan ◽  
L Gerace
Keyword(s):  
Cdna Cloning ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Coiled Coil ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Repeat Region ◽  
Transport Activity ◽  
Coiled Coil Region ◽  
Gated Channel

Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.

scholarly journals Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.

1995 ◽  
Vol 6 (11) ◽  
pp. 1591-1603 ◽  
Author(s):  
T Guan ◽  
S Müller ◽  
G Klier ◽  
N Panté ◽  
J M Blevitt ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Transport Channel ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Gated Channel

The p62 complex is an oligomeric assembly of O-linked glycoproteins of the nuclear pore complex that interacts with cytosolic transport factors and is part of the machinery for nuclear protein import. In this study we have purified the p62 complex from rat liver nuclear envelopes and analyzed its structure and composition. The p62 complex consists of four distinct polypeptides (p62, p58, p54, and p45) and has a mass of approximately 234 kDa, calculated from its hydrodynamic properties and supported by chemical cross-linking and scanning transmission electron microscopy. These data suggest that the p62 complex contains one copy of each constituent polypeptide. Analysis of preparations of the p62 complex by electron microscopy using rotary metal shadowing and negative staining revealed donut-shaped particles with a diameter of approximately 15 nm. Immunogold electron microscopy of isolated rat liver nuclear envelopes demonstrated that p62 occurs on both the nucleoplasmic and cytoplasmic sides of the pore complex near the central gated channel involved in active transport of proteins and RNAs. The properties and localization of the p62 complex suggest that it may be involved in binding transport ligands near the center of the nuclear pore complex and in subsequently transferring them to the gated transport channel.

A biochemical and immunological comparison of nuclear and cytoplasmic pore complexes

1996 ◽  
Vol 109 (7) ◽  
pp. 1813-1824 ◽  
Author(s):  
A. Ewald ◽  
U. Kossner ◽  
U. Scheer ◽  
M.C. Dabauvalle
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Transport Processes ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Surface ◽  
Annulate Lamellae ◽  
Binding Studies ◽  
Pore Complexes

Pore complexes are not confined to the nuclear envelope but can also be found in the cytoplasm of numerous cell types in the form of annulate lamellae (AL). We have induced formation of AL by exposure of rat cells (line RV) to sublethal doses of the antimitotic drug vinblastine sulfate, and compared the distribution of several nuclear pore complex proteins (nucleoporins) in the nuclear envelope and AL by immunocytochemistry, cytochemical lectin binding studies and immunoblot analyses of nuclear and AL-enriched fractions. All the antibodies used yielded punctate nuclear surface staining in immunofluorescence microscopy which is characteristic for nuclear pore complex components. When we applied antibodies against the nucleoporin p62, AL were visualized as numerous cytoplasmic dot-like structures. Immunogold electron microscopy confirmed the correspondence of the cytoplasmic bodies with stacks of AL. Antibodies to constituents of the cytoplasmic (nup180) and nucleoplasmic (nup153) filaments extending from both sides of nuclear pore complexes also stained the AL, indicating that pore complexes are intrinsically asymmetric assemblies independent of their specific intracellular topology. By contrast, AL were negative with five different antibodies against the transmembrane nuclear pore glycoprotein gp210 and the lectin concanavalin A (ConA) known to bind to the oligosaccharide side chains of gp210. Similarly, there was no staining of the AL with antibodies to the other nuclear pore membrane protein so far known in higher eukaryotes, POM121. Immunoblot analyses confirmed the presence of p62, nup180 and nup153 in both the nuclear and AL fractions and the absence of gp210 and POM121 from AL. Our results do not support the generally held view that gp210 and POM121 function in anchoring the pore complex scaffold to the pore membrane. Rather, they point to a role for these proteins in transport processes through the nuclear pore complexes. Since AL are not involved in nucleocytoplasmic transport processes they may lack components of the transport machinery.

scholarly journals Antigen Retrieval Trial for Post-embedding Immunoelectron Microscopy by Heating with Several Unmasking Solutions

2003 ◽  
Vol 51 (8) ◽  
pp. 989-994 ◽  
Author(s):  
Nagahito Saito ◽  
Kohei Konishi ◽  
Hiroshi Takeda ◽  
Mototsugu Kato ◽  
Toshiro Sugiyama ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Phosphate Buffer ◽  
Tris Buffer ◽  
Antigen Retrieval ◽  
Immunogold Electron Microscopy ◽  
Buffer Solution ◽  
Microscopy Method ◽  
Ultrathin Sections ◽  
H Pylori ◽  
Lowicryl K4m

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti- H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in postembedding immunogold electron microscopy.

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