Alpha-spectrin in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts.
The distribution of alpha-spectrin, and its relation to other cytoskeletal structures and to the plasma membrane, was studied in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts by using immunogold labelling and electron microscopy (IEM). The cell surface was labelled with gold-conjugated wheat germ agglutinin (WGA-gold), microtubules with anti-tubulin antibodies, and spectrin by using antibodies raised to chicken erythrocyte alpha-spectrin. Additionally, the effect of fixation and drying on the labelling pattern was evaluated. In electron microscopy, a three-dimensional filamentous network was observed in detergent-extracted whole-mount preparations. Filaments of diameter 7-10 nm and 15 nm, microtubules of diameter 30 nm, and filament bundles (40-50 nm in diameter) were seen. In IEM, alpha-spectrin was seen on the surface of the cytoskeletal network, especially along the thick filament bundles. In some cells, a distinct membrane skeleton which was labelled with alpha-spectrin antibodies, was seen in close association with the cytoskeletal network. The cells which were labelled first with WGA-gold, and then permeabilized, fixed and labelled with alpha-spectrin, showed a co-localization of the WGA binding sites and alpha-spectrin along the surface of the filament bundles. Reversing the order of the staining, such that fixation was done before WGA labelling and permeabilization, led to a greatly diminished labelling for alpha-spectrin and less pronounced co-localization of spectrin and WGA. Comparison of the conventional critical point drying method with Peldri II, a novel drying agent, indicated a better stability of the cellular structures under the electron beam when Peldri II was used.(ABSTRACT TRUNCATED AT 250 WORDS)
Sormunen
R
R
Biocenter, University of Oulu, Finland.
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002642:Chick Embryo; D003599:Cytoskeleton / Q000737:chemistry* / Q000648:ultrastructure; D003902:Detergents; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology / Q000648:ultrastructure; D013049:Spectrin / Q000032:analysis*; D014909:Wheat Germ Agglutinins
D000911:Antibodies, Monoclonal; D003902:Detergents; D014909:Wheat Germ Agglutinins; D013049:Spectrin