Abstract. Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment l, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction.The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to a-actinin: a-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with a-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to a-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

No abstract

Monospecific antibodies to chicken gizzard actin, c~-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 c~-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 c~-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-c~-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.The motile processes of cultured cells, such as the formation of cellular contacts, cell spreading, shape changes, cell retraction, and locomotion, ruffling, surface motility, and cytokinesis, are to a large extent mediated by F-actin microfilaments (for reviews see references 4,19,26,48,61). The microfilament system in cultured cells occurs in a few characteristic patterns in the different cell domains (4,5,18,28,46): (a) In the leading edge--a highly organized three-dimensional network adjacent to parallel arrays of microspikes and filopodia; (b) in the cell body--(/) networks or mats of microfilaments differing in degree of organization and density, mainly confined to the cell cortex and (ii) prominent and densely packed microfilament bundles or "stress fibers," their termini, and polygonal nets. 1324By the use of light microscopic antibody localizations (reviewed in reference 25) and microinjection studies, it has become apparent that a number of mechanochemical proteins, including a-actinin (13,23, 38), filamin (54), tropomyosin (36, 37, 59), myosin (14, 56, 65), fimbrin (3), and vinculin (15), are associated with this actin matrix. Moreover, their characteristic distribution patterns observed ...

No abstract

No abstract