Identification and characterization of a nuclear pore complex protein

Davis, Laura I.; Blobel, Günter

doi:10.1016/0092-8674(86)90784-1

Cited by 596 publications

(441 citation statements)

References 26 publications

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“…After high ionic strength salt extraction, punctate fluorescence was still detected, but there was a 50% decrease in intensity ( Figure If). This reactivity profile of serum LL-reactive antigens was similar to that reported for nuclear pore complex proteins (14,15).…”

Section: Resultssupporting

confidence: 86%

“…These proteins were enriched in nuclei and in nuclear envelope preparations. Treatment of these envelopes with 0.5M NaCl diminished the labeling of these bands (Figure 3), as was expected from the immunofluorescence results, and the solubility of known nuclear pore complex proteins (14). Incubation of nuclear envelopes with a buffer known to extract nuclear pore complexes abolished labeling of these proteins ( Figure 3).…”

Section: Resultssupporting

confidence: 70%

“…These proteins are distinct from the 62-72 kd range of nuclear lamins described in BHK-21 cells (10). Partial solubilization of these polypeptides occurred with NaCl treatment, as observed in the immunofluorescence studies, and as reported by Davis and Blobel for polypeptide components of nuclear pore complexes (14). Recently, different nuclear pore complex proteins have been described (1616).…”

Section: Discussionmentioning

confidence: 66%

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A novel autoantibody causing a peripheral fluorescent antinuclear antibody pattern is specific for nuclear pore complexes

Dagenais

Bibor-Hardy

Senécal

1988

Arthritis & Rheumatism

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We characterized serum from a patient with polymyositis, and found that it produced a peripheral (rim) fluorescent antinuclear antibody pattern on rat liver substrate. Indirect immunofluorescence analysis revealed a punctate pattern at the nuclear surface of PtK2, BHK-21, and HEp-2 cells. This pattern was still present after sequential extraction in situ with non-ionic detergent, DNase, RNase, and high ionic strength buffer (2M NaCI). Immunogold electron microscopic localization was specific for nuclear pore complexes. By immunoblot analysis, the antigens were polypeptides of 200 kd and 130 kd that were enriched in the nuclear fraction.Antinuclear antibodies (ANA) in human sera are routinely detected with indirect immunofluorescence microscopy on substrates such as rat liver sections (1). Among the patterns identified by this method, the peripheral (rim) pattern is of diagnostic importance because of its association with systemic lupus erythematosus (SLE) (1,2). Studies of the antigenic specificity of autoantibodies from sera causing a peripheral ANA pattern have shown a strong association with anti-DNA antibodies (2,3). In addition, autoantibodies to nuclear lamins, observed in sera from 1 patient with linear scleroderma (4) and 5 patients with SLE (5,6), were shown to cause a peripheral ANA pattern because of continuous labeling of the nuclear envelope.Nuclear pore complexes are structures localized at the points of fusion of the double nuclear membranes (7) and are thought to allow molecular exchanges across the nuclear envelope. We report the identification, in serum from a patient with polymyositis, of a novel autoantibody that causes a peripheral ANA pattern on rat liver sections and stains the nuclear envelope of various cell lines. This autoantibody reacts with nuclear pore complexes. PATIENTS AND METHODSPatient LL. The index serum was obtained from a 35-year-old white woman who, in December 1985, developed polymyositis, which was characterized by progressive proximal muscle weakness, elevated levels of serum creatine kinase (4,836 units/liter, normal 24-1 70), myopathic changes evident on electromyogram, and abnormal findings on quadriceps muscle biopsy. ANA were detected by indirect immunofluorescence on cryostat rat liver sections, at a titer of 1: 1,280 and with a peripheral pattern. Esophageal manometry revealed decreased peristaltic pressure in the distal two-thirds of the esophagus.Normal or negative results were noted on the following tests: hemoglobin, leukocyte and platelet

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Section: Resultssupporting

confidence: 86%

Section: Resultssupporting

confidence: 70%

Section: Discussionmentioning

confidence: 66%

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A novel autoantibody causing a peripheral fluorescent antinuclear antibody pattern is specific for nuclear pore complexes

Dagenais

Bibor-Hardy

Senécal

1988

Arthritis & Rheumatism

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“…Cells were grown to early log phase in YPD liquid at 23°C and were shifted to 37°C for varying times before fixation. Fixed cells were incubated with the appropriate antibody [undiluted tissue culture supernatant (tcs) MAb D77 (Henriquez et al, 1990) for detection of Noplp; 1:100 diluted tcs MAb 5B5 (gift from M. Rout and J. Kilmartin) for detection of Nsrlp; 1:50 diluted anti-f3-galactosidase MAb from Boehringer Mannheim (Mannheim, Germany); undiluted tcs MAb 12CA5 (Berkeley Antibody, Berkeley, CA) for detection of HAGle2p; undiluted tcs MAb 414 (Davis and Blobel, 1986) for detection of NPCs] for 16 h at 4°C. After washing with 40 mM K2HPO4, 10 mM KH2PO4, 150 mM NaCl, 0.1% NaN3, 0.1% Tween 20, and 2% nonfat dry milk (M buffer) alone, detection of bound antibody was accomplished by incubation with affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel Laboratories, Organon Teknika, Durham, NC) at a 1:100 dilution for 1 h at room temperature.…”

Section: Immunofluorescence and Electron Microscopymentioning

confidence: 99%

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

Murphy

Watkins

Wente

1996

MBoC

168

164

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To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin NuplOOp were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srplp, the nuclear localization signal receptor, as well as Riplp, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

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“…Cells were permeabilized with prechilled 0.5% Triton X in PBS or 40 g/ml digitonin (SigmaAldrich) in PBS for a total of 15 min, blocked with 5% milk in PBS for 30 min, and then washed three times with the same solution used in permeabilizing step (Adam et al, 1992). Permeabilized cells were then incubated with the appropriate primary antibodies for 1 h: undiluted mouse anti-UNC-83 monoclonal antibody (mAb) (as described above), rat anti-UNC-84 antibody used at a dilution of 1/1000 (as described above), mouse anti-nuclear pore complex monoclonal 414 antibody used at a dilution of 1/1000 (Covance) (Davis and Blobel, 1986), and goat anti-lamin B antibody (Santa Cruz Biotechnology, Santa Cruz, CA) used at a dilution of 1/100. After rinsing the cells three times with permeabilization buffer, a 1-h incubation with the appropriate secondary antibodies followed: donkey anti-mouse antibody conjugated to Cy3, donkey anti-rat antibody conjugated to Cy3, or donkey anti-goat antibody conjugated to Cy2 (Jackson ImmunoResearch Laboratories).…”

mentioning

confidence: 99%

UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

McGee

Rillo

Anderson

et al. 2006

MBoC

127

180

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UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane. INTRODUCTIONA variety of cellular and developmental processes, including fertilization, cell division, cell migration, and establishment of polarity, depend on positioning the nucleus to a specific location within the cell. For example, in budding yeast the nucleus must migrate to the bud neck before the onset of mitosis. Also, nuclei actively follow the leading edge of migratory cells, such as those in the developing cerebral cortex. Nuclear migration defects in these two examples lead to the missegregation of chromosomes or the neurological disease lissencephaly, respectively (reviewed in Morris, 2000). The role of microtubules and associated dynein and kinesin motors in nuclear migration are well established (reviewed in Reinsch and Gonczy, 1998). Although less established, actin also plays an important role in many nuclear positioning events (reviewed in Starr and Han, 2003). Recently, a definitive role for actin networks has been described in nuclear migration during NIH 3T3 cell polarization (Gomes et al., 2005). It remains relatively unknown how the nucleus connects to the cytoplasmic cytoskeleton during nuclear migration. Furthermore, it is not clear how the forces involved in nuclear positioning are transferred across both membranes of the nuclear envelope from the cytoskeleton to the nuclear matrix.We have previously proposed that two C. elegans proteins, UNC-84 and UNC-83, function to control nuclear migration by bridging the nuclear envelope, connecting the cytoskeleton with the nuclear matrix (Starr et al., 2001;Lee et al., 2002;Starr and Han, 2003). Mutations in unc-83 or unc-84 disrupt nuclear migration in at least three cell types: embryonic hypodermal hyp7 precursors, larval hypodermal P-cells, and embryonic intestinal primordial cells (Horvitz and Sulst...

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Identification and characterization of a nuclear pore complex protein

Cited by 596 publications

References 26 publications

A novel autoantibody causing a peripheral fluorescent antinuclear antibody pattern is specific for nuclear pore complexes

A novel autoantibody causing a peripheral fluorescent antinuclear antibody pattern is specific for nuclear pore complexes

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

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