Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method
| Autor: | G. Langanger, Guy Daneels, J. De Mey, M. Moeremans, M. De Brabander, J.V. Small |
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| Rok vydání: | 1984 |
| Předmět: |
Filamins
Fluorescent Antibody Technique Chick Embryo macromolecular substances Actinin Biology Microfilament Filamin Antibodies Epithelium Contractile Proteins Animals Lung Actin Staining and Labeling Myocardium Microfilament Proteins Myosin Subfragments Muscle Smooth Articles Cell Biology Immunogold labelling Microfilament Protein Peptide Fragments Cell biology Staining Microscopy Electron Actinin alpha 1 Gizzard Avian Gold Carrier Proteins |
| Zdroj: | The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| DOI: | 10.1083/jcb.99.4.1324 |
| Popis: | Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system. |
| Databáze: | OpenAIRE |
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