Five chemically distinct classes of intermediate filaments can be identified within higher eukaryotic cells. Each class is characteristic of a particular cell type. These filaments may function to integrate mechanically the various structures of the cytoplasmic space in a way that is tailored to the differentiated state of the cell.

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Antibodies specific for the skeletal muscle structural protein alpha-actinin are used to localize this protein by indirect immunofluorescence in nonmuscle cells. In cultured nonmuscle cells, alpha-actinin is localized along or between actin filament bundles producing an almost regular periodicity. The protein is also detected in the form of fluorescent plaques at some ends of actin filament bundles, as well as in a filamentous form in some overlap areas of cells. In spreading rat embryo cells, alpha-actinin assumes a focal distribution which corresponds to the vertices of a highly regular actin filament network. The results suggest that alpha-actinin may be involved in the organization of actin filament bundles, in the attachment of actin filaments to the plasma membrane, and in the assembly of actin filaments in areas of cell to cell contact.

Actin purified from mouse fibroblasts by sodium dodecyl sulfate gel electrophoresis was used as antigen to obtain an antibody in rabbits. The elicited antibody was shown to be specific for actin as judged by immunodiffusion and complement fixation against partially purified mouse fibroblast actin and highly purified chicken muscle actin. The antibody was used in indirect immunofluorescence to demonstrate by fluorescence light microscopy the distribution and pattern of actin-containing filaments in a variety of cell types. Actin filaments were shown to span the cell length or to concentrate in "focal points" in patterns characteristic for each individual cell.Eucaryotic cells contain three basic fibrous structures: filaments, microfilaments, and microtubules. These three structures are thought to be intimately involved in the maintenance of cell shape, in cell movement, and in other important cellular functions (1). Microfilaments are thought to contain actin. This assumption is based on the observation that these structures can be selectively decorated with heavy meromyosin, a specific proteolytic fragment of muscle myosin known to interact with muscle actin (2). Furthermore, actin is now now known to exist as a major protein component of a variety of non-muscle cellular types and in each case it has properties markedly similar to those of its muscle counterpart (3-7)*. The major protein of the microtubular system, tubulin, has been isolated and well characterized (9). The basic protein subunit of the filament structure, however, has not so far been identified. Presumptive muscle proteins like myosin (10)(11)(12)(13) and tropomyosin (14) have been found in some non-muscle cells. However, their exact distribution within the cell, as well as their specific localization in one of these fibrous structures, is as yet undetermined.We have developed a relatively simple way of selectively visualizing filamentous structures in the cell by using antibodies made against different structural proteins. was used to obtain an antibody in rabbits. The antiserum obtained was shown by immunodiffusion and complement fixation to be specific for actin. This antibody was then used in indirect immunofluorescence to show that microfilaments are polymers of actin. This technique also enabled us to demonstrate the complex network of actin filaments in a variety of cell types. MATERIALS AND METHODSGrowth of Cells. Actin was isolated from the cell line SV101, a clone of mouse fibroblast 3T3 cells transformed by Simian virus 40. This transformed cell line was chosen because it grows to a higher saturation density than the parent 3T3 cell line (15). The cells were grown in roller bottles (Vitro Corp.) in Dulbecco's modified Eagle's medium containing 10% calf serum and 50 ug/ml of gentamycin. At confluency, the medium was removed and the cells were washed with phosphatebuffered saline (PBS). The cells were then scraped off the bottles, collected by low speed centrifugation, and stored at -70gPreparation of Actin. The cells were thawed...

Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3.1.4.5). In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle The observation that actin can be isolated by DNaseagarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions.