The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborizea or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transfe-med cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp6src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.Cytochalasin B (CB) inhibits a variety of cell functions associated with metabolite transport, cell movement, and morphology (29, 30). Fibroblasts exposed to CB retract their cytoplasm, leaving behind long dendritic-like processes. This morphological transition has been termed "arborization" (11,20,39). CB affects the integrity of the actin-containing plasmalemmal microfilaments. Electron microscopy and immunofluorescence studies have shown that, in CB, the extensive microfilament network of fibroblasts is broken down and replaced by short oligomers (37; see below). The effect of CB on the polymerization of microfilaments appears to be due to its binding to the rapidly polymerizing end of the actin filaments (8,14,31) ing, the determination of cellular morphology, and cell movement. Many of these cellular functions are altered after transformation of normal chicken embryo fibroblasts with Rous sarcoma virus (RSV). It has been suggested that the alteration of microfilaments which often characterizes the transformed phenotype (12,35,40,42,45)

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallelaligned intermediate filaments. Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41°C). At the permissive temperature for transformation (36°C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41°C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.When trypsinized fibroblasts are plated in culture, they attach to and spread on the substrate within several hours. This spreading process involves cell movement and is accompanied by the establishment of an actin-containing microfilament network within the cytoplasm just under the plasma membrane. These microfilaments are implicated as an essential element in cell spreading (13) and are required for many cell movements, including cytokinesis and translational movement. The extracellular matrix proteins are another major element in cell spreading, particularly fibronectin, which is laid down as a network on the substrate under the spreading cell (see reference 33 for a review). There are conflicting data on the association of the fibronectin component of the extracellular matrix with the microfilaments and the microfilamentassociated proteins. One line of evidence indicates that the fibronectin matrix is present all along the plasma membrane leading up to but not including the area of adhesion plaques (5), whereas another group of investigators have evidence of a transmembrane linkage of fibronectin and vinculin fibers at the adhesion plaques (27). These fibronectin-vinculin complexes are aligned with the microfilament bundles found along the substrate within the cell.The role of fibronectin in cell spreading is demonstrated by the ability of exogenously added fibronectin to increase the spreading of poorly spread, transformed cells (34). Also, the addition of fibronectin to chondroblast floater cells induces both attachment and spreading of the cells (32). Cooperative effects of actin and fibronectin in the det...