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.

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 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.

Mass determination of the nuclear pore complex and its sub-complexesl by high-resolution Scanning Transmission Electron Microscopy (Stem)

1989 ◽  
Vol 47 ◽  
pp. 814-815
Author(s):  
R. Reichelt ◽  
A. Holzenburg ◽  
A. Engel ◽  
U. Aebi
Keyword(s):  
High Resolution ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Carbon Film ◽  
Nuclear Pore ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Thin Arrow ◽  
Thick Arrow

The nuclear pore complex (NPC) is an elaborate membrane-bound molecular machine residing in the nuclear envelope of all eukaryotic cells and acting as a passageway for molecular transport between the nucleus and the cytoplasm. Complementary to transmission electron microscopic studies of negatively stained and frozen hydrated NPCs yielding detailed information about their overall size, shape and substructure, the high-resolution scanning transmission electron microscope (STEM) was used to measure the mass, to map the radial mass distribution, and to evaluate the rotational symmetry of unstained native NPCs and of several distinct sub-complexes. “Native” NPCs were prepared for STEM as follows: the nucleus of Xenopus laevis oocytes was placed on a 4 nm carbon film supported by a holey film mounted on a copper mesh grid, and its nuclear envelope ruptured and spread out using fine glass needles. The grid was washed with low salt buffer to remove macroscopic contaminants, and excess liquid was withdrawn with filter paper. Next, tobacco mosaic virus (TMV; serves as internal mass standard) was co-adsorbed, before the grid was washed for 10 sec each on 3 drops of bi-destilled H2O. To disintegrate the NPC into distinct sub-complexes, nuclei were placed on a 4-nm thick carbon film supported by a thick holey carbon foil on a copper grid, ruptured and spread as above, and then detergent extracted. After washing, the samples were frozen in liquid N2, transferred to a VG HB-5 STEM and freeze-dried at 153 K. Low-dose annular dark field (AD) images (50 to 300 e/nm2) were acquired digitally, corrected for nonlinearities in the AD signal due to high mass thickness, and stored on tape for further processing.Native NPCs, with and without plug, are displayed in Fig. 1. As shown in Fig. 2, two types of rings can be distinguished after detergent extraction: bright rings (“outer” rings; thick arrow) and dimmer rings (“inner” rings; thin arrow). In the same preparations particle “pairs” consisting of a ring (Fig. 3, thin arrow) and a plug-spoke complex (Fig. 3, thick arrow) can often be depicted. From these data averaged and 8-fold symmetrized mass maps of the native NPC with plug (Fig. 4; ˜50 NPCs) and without plug (Fig. 5; ˜50 NPCs), the outer ring (Fig. 6; ˜50 rings) and the inner ring (Fig. 7; ˜15 rings) were computed. Corresponding radial mass distributions are displayed at the same scale for comparison (Figs. 8 to 11). The average masses of the different structures are summarized in Table 1 together with a tentative model. These measurements bring speculations about the mass of the NPC to an end which have stated values ranging from 20 MDa to over 100 MDa, and they suggest that the NPC could easily be made of the order of 100 different polypeptides.

Steps of nuclear pore complex disassembly and reassembly during mitosis in earlyDrosophilaembryos

2001 ◽  
Vol 114 (20) ◽  
pp. 3607-3618 ◽  
Author(s):  
Elena Kiseleva ◽  
Sandra Rutherford ◽  
Laura M. Cotter ◽  
Terence D. Allen ◽  
Martin W. Goldberg
Keyword(s):  
Electron Microscopy ◽  
Nuclear Pore Complex ◽  
Drosophila Embryo ◽  
Nuclear Pore ◽  
Polar Regions ◽  
Transmission Electron ◽  
Vitro Model ◽  
First Time

The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional ‘spindle envelope’. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.

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 Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy

1993 ◽  
Vol 122 (1) ◽  
pp. 1-19 ◽  
Author(s):  
CW Akey ◽  
M Radermacher
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Three Dimensional ◽  
Nucleocytoplasmic Transport ◽  
Domain Model ◽  
Nuclear Pore ◽  
Transport Channel ◽  
Macromolecular Transport ◽  
Cryo Electron Microscopy

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent-extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.

High-Voltage Scanning Electron Microscopy

1970 ◽  
Vol 28 ◽  
pp. 6-7
Author(s):  
J. M. Cowley
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Scanning Transmission Electron Microscopy ◽  
Wave Optics ◽  
Contrast Effects ◽  
Transmission Electron ◽  
Mode Of Operation ◽  
Scanning Transmission ◽  
Types Of Information ◽  
Voltage Scanning

The comparison of scanning transmission electron microscopy (STEM) with conventional transmission electron microscopy (CTEM) can best be made by means of the Reciprocity Theorem of wave optics. In Fig. 1 the intensity measured at a point A’ in the CTEM image due to emission from a point B’ in the electron source is equated to the intensity at a point of the detector, B, due to emission from a point A In the source In the STEM. On this basis it can be demonstrated that contrast effects In the two types of instrument will be similar. The reciprocity relationship can be carried further to include the Instrument design and experimental procedures required to obtain particular types of information. For any. mode of operation providing particular information with one type of microscope, the analagous type of operation giving the same information can be postulated for the other type of microscope. Then the choice between the two types of instrument depends on the practical convenience for obtaining the required Information.

Development of 200 kV Scanning-Transmission Electron Microscope

1974 ◽  
Vol 32 ◽  
pp. 410-411
Author(s):  
H. Koike ◽  
S. Sakurai ◽  
K. Ueno ◽  
M. Watanabe
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Scanning Transmission Electron Microscope ◽  
The Other ◽  
Morphological Observation ◽  
Magnetic Domains ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Backscattered Electron ◽  
Increasing Demand

In recent years, there has been increasing demand for higher voltage SEMs, in the field of surface observation, especially that of magnetic domains, dislocations, and electron channeling patterns by backscattered electron microscopy. On the other hand, the resolution of the CTEM has now reached 1 ∼ 2Å, and several reports have recently been made on the observation of atom images, indicating that the ultimate goal of morphological observation has beem nearly achieved.

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