Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method.
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.
D000185:Actinin / Q000032:analysis*; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000032:analysis*; D002642:Chick Embryo; D003285:Contractile Proteins / Q000032:analysis*; D004848:Epithelium / Q000648:ultrastructure; D064448:Filamins; D005455:Fluorescent Antibody Technique; D005895:Gizzard, Avian / Q000648:ultrastructure; D006046:Gold; D008168:Lung / Q000648:ultrastructure*; D008840:Microfilament Proteins / Q000032:analysis*; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000648:ultrastructure*; D009206:Myocardium / Q000648:ultrastructure*; D015879:Myosin Subfragments / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D013194:Staining and Labeling
D000906:Antibodies; D002352:Carrier Proteins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000185:Actinin; D006046:Gold