Quantitative immunogold localization of protein phosphatase 2B (Calcineurin) in Paramecium Cells

Lade...
Vorschaubild
Datum
2000
Autor:innen
Herausgeber:innen
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
ArXiv-ID
Internationale Patentnummer
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Open Access Green
Sammlungen
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published
Erschienen in
Zusammenfassung

For immunogold EM labeling analysis, we fixed Paramecium cells in 4% formaldehyde and 0.125% glutaraldehyde, followed by low-temperature embedding in unicryl and UV polymerization. We first quantified some obvious but thus far neglected side effects of section staining on immunogold labeling, using mono- or polyclonal antibodies (Abs) against defined secretory and cell surface components, followed by F(ab)2 or protein A gold conjugates. Use of alkaline lead staining resulted in considerable rearrangement and loss of label unless sections were postfixed by glutaraldehyde after gold labeling. This artifact is specific for section staining with lead. It can be avoided by staining sections with aqueous uranyl acetate only to achieve high-resolution immunogold localization of a protein phosphatase on unicryl sections. In general, phosphatases are assumed to be closely, although loosely, associated with their targets. Because the occurrence of protein phosphatase 2B (calcineurin) in Paramecium has been previously established by biochemical and immunological work, as well as by molecular biology, we have used Abs against mammalian CaN or its subunits, CaN-A and CaN-B, for antigen mapping in these cells by quantitative immunogold labeling analysis. Using ABs against whole CaN, four structures are selectively labeled (with slightly decreasing intensity), i.e., infraciliary lattice (centrin-containing contractile cortical filament network), parasomal sacs (coated pits), and outlines of alveolar sacs (subplasmalemmal calcium stores, tightly attached to the cell membrane), as well as rims of chromatin-containing nuclear domains. In other subcellular regions, gold granules reached densities three to four times above background outside the cell but there was no selective enrichment, e.g., in cilia, ciliary basal bodies, cytosol, mitochondria, trichocysts (dense-core secretory organelles), and non-chromatin nuclear domains. Their labeling density was 4- to 8.5-fold (average 6.5-fold) less than that on selectively labeled structures. Labeling tendency was about the same with Abs against either subunit. Our findings may facilitate the examination of molecular targets contained in the selectively labeled structure.

Zusammenfassung in einer weiteren Sprache
Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
calcineurin, ciliates, immunolocalization, Paramecium, phosphatase 2B, protozoa
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690MOMAYEZI, Massoud, Roland KISSMEHL, Helmut PLATTNER, 2000. Quantitative immunogold localization of protein phosphatase 2B (Calcineurin) in Paramecium Cells. In: Journal of Histochemistry and Cytochemistry. 2000, 48(9), pp. 1269-1281. ISSN 0022-1554. eISSN 1551-5044. Available under: doi: 10.1177/002215540004800910
BibTex
@article{Momayezi2000Quant-7819,
  year={2000},
  doi={10.1177/002215540004800910},
  title={Quantitative immunogold localization of protein phosphatase 2B (Calcineurin) in Paramecium Cells},
  number={9},
  volume={48},
  issn={0022-1554},
  journal={Journal of Histochemistry and Cytochemistry},
  pages={1269--1281},
  author={Momayezi, Massoud and Kissmehl, Roland and Plattner, Helmut}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/7819">
    <dc:rights>Attribution-NonCommercial-NoDerivs 2.0 Generic</dc:rights>
    <dc:creator>Momayezi, Massoud</dc:creator>
    <dc:contributor>Plattner, Helmut</dc:contributor>
    <dcterms:rights rdf:resource="http://creativecommons.org/licenses/by-nc-nd/2.0/"/>
    <dc:contributor>Kissmehl, Roland</dc:contributor>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:37:46Z</dc:date>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7819/1/Quantitative_immunogold_localization_of_protein_phosphatase_2B.pdf"/>
    <dc:contributor>Momayezi, Massoud</dc:contributor>
    <dc:language>eng</dc:language>
    <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/7819"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7819/1/Quantitative_immunogold_localization_of_protein_phosphatase_2B.pdf"/>
    <dcterms:abstract xml:lang="eng">For immunogold EM labeling analysis, we fixed Paramecium cells in 4% formaldehyde and 0.125% glutaraldehyde, followed by low-temperature embedding in unicryl and UV polymerization. We first quantified some obvious but thus far neglected side effects of section staining on immunogold labeling, using mono- or polyclonal antibodies (Abs) against defined secretory and cell surface components, followed by F(ab)2  or protein A gold conjugates. Use of alkaline lead staining resulted in considerable rearrangement and loss of label unless sections were postfixed by glutaraldehyde after gold labeling. This artifact is specific for section staining with lead. It can be avoided by staining sections with aqueous uranyl acetate only to achieve high-resolution immunogold localization of a protein phosphatase on unicryl sections. In general, phosphatases are assumed to be closely, although loosely, associated with their targets. Because the occurrence of protein phosphatase 2B (calcineurin) in Paramecium has been previously established by biochemical and immunological work, as well as by molecular biology, we have used Abs against mammalian CaN or its subunits, CaN-A and CaN-B, for antigen mapping in these cells by quantitative immunogold labeling analysis. Using ABs against whole CaN, four structures are selectively labeled (with slightly decreasing intensity), i.e., infraciliary lattice (centrin-containing contractile cortical filament network), parasomal sacs (coated pits), and outlines of alveolar sacs (subplasmalemmal calcium stores, tightly attached to the cell membrane), as well as rims of chromatin-containing nuclear domains. In other subcellular regions, gold granules reached densities three to four times above background outside the cell but there was no selective enrichment, e.g., in cilia, ciliary basal bodies, cytosol, mitochondria, trichocysts (dense-core secretory organelles), and non-chromatin nuclear domains. Their labeling density was 4- to 8.5-fold (average 6.5-fold) less than that on selectively labeled structures. Labeling tendency was about the same with Abs against either subunit. Our findings may facilitate the examination of molecular targets contained in the selectively labeled structure.</dcterms:abstract>
    <dcterms:bibliographicCitation>First publ. in: Journal of Histochemistry and Cytochemistry 48 (2000), 9, pp. 1269-1281</dcterms:bibliographicCitation>
    <dc:creator>Kissmehl, Roland</dc:creator>
    <dc:format>application/pdf</dc:format>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:37:46Z</dcterms:available>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dcterms:issued>2000</dcterms:issued>
    <dcterms:title>Quantitative immunogold localization of protein phosphatase 2B (Calcineurin) in Paramecium Cells</dcterms:title>
    <dc:creator>Plattner, Helmut</dc:creator>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Nein
Begutachtet
Diese Publikation teilen