Double immunofluorescence staining of quail embryo fibroblasts with rabbit antibody to vinculin and mouse monoclonal antibody to vimentin revealed a coincidence between fluorescence patterns for cell-substrate focal contacts and intermediate filaments. Most of the vinculin-containing adhesion plaques coincided with the ends of vimentin-positive fibrils. This association was further corroborated by immunoelection microscopic observations of the cytoskeletons of quail and mouse fibroblasts using a platinum replica technique. The intermediate filaments were identified either by direct treatment with antivimentin IgM or by an indirect immunogold staining method. Colcemid treatment of the cells caused a collapse of intermediate filaments and destroyed their association with focal contacts. During the early stages of the colcemid-induced collapse of the intermediate filaments, single vimentin fibrils appeared to retain their association with focal contacts. The possible role of the intermediate filaments in the formation and maintenance of focal contacts is discussed.

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Normal cultured mouse fibroblasts spreading on solid substrate extend and attach numerous pseudopods; lamellar cytoplasm is eventually formed from the attached pseudopods. Fibroblasts spreading in the presence of cytochalasin B (CB) form de novo a system of arbor-like branched processes rather than lamellar cytoplasm. The Spreading of cultured fibroblasts on various solid substrates is a convenient process for investigation of mechanisms of cell shape alterations and of cell attachment to noncellular surfaces. Morphology of spreading had been studied in detail (see review in ref. 1). It has been shown that spreading is a result of extension and attachment of numerous pseudopods. These pseudopodial reactions eventually lead to formation of special substrate-attached structure, lamellar cytoplasm. Pseudopods and lamellar cytoplasm formed in the course of spreading are highly contractile; they also have the distinctive ability to clear their surfaces from the patches of receptors crosslinked by a corresponding ligand (2, 3). Numerous microfilament bundles are formed in the course of spreading; the ends of these bundles are associated with focal cell-substrate contacts (4-6). Radial growth of microtubules into the lamellar cytoplasm is another characteristic feature of spreading (7).To analyze further the cellular reactions involved in spreading we have examined the alterations in the course of this process caused by cytochalasin B (CB). The effects of CB on the fully spread fibroblasts are well known: it destroys partially or completely the structures consisting of actin microfilaments, it inhibits pseudopodial activity and other cellular movements, and it transforms lamellar cytoplasm into an arbor-like structure consisting of branched cytoplasmic processes (8-11). As first shown by Goldman and Knipe (12), CB does not completely stop the spreading of fibroblasts: a cell can form de novo an arbor-like structure in the presence of this inhibitor. In the experiments described below we compared the properties of pseudopods and of arbor-like structures formed in the presence of CB with those of the structures formed in the course of normal spreading.The results of our experiments give reason to suggest that extension and attachment of pseudopods can be dissociated 5919 from the processes that make these pseudopods highly contractile and able to clear their surfaces from patched receptors. MATERIALS AND METHODSCultures of normal mouse fibroblasts were obtained by trypsinization of minced embryos taken at 15-16 day of pregnancy. The cells were grown in stoppered glass flasks in a mixture of equal parts of Eagle's medium and of 0.5% lactalbumin hydrolysate to which 10% bovine serum was added. The cells from primary cultures were removed from the glass by trypsinization and suspended either in the usual medium (control medium) or in medium containing 10 mg of CB per ml (CB medium). The cells in control or in CB medium were then placed in glass flask; if necessary, a glass coverslip was placed at the bottom of the...