Altered Cell Spreading in Cytochalasin B: a Possible ... - Europe PMC

Vol. 3, No. 1

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1983, p. 113-125 0270-7306/83/010113-13$02.00/0 Copyright © 1983, American Society for Microbiology

Altered Cell Spreading in Cytochalasin B: a Possible Role for Intermediate Filaments A. S. MENKO,l* Y. TOYAMA,2 D. BOETTIGER,3 AND H. HOLTZER2 Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108,' and Departments of Anatomy2 and Microbiology,3 University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received 8 March 1982/Accepted 6 October 1982

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. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a 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 transla-

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 determination of cell shape have been suggested by double immunofluorescence labeling experiments which demonstrate that the fibronectin fibers are coincident with microfilament bundles within the cell (15, 17, 26). It has been postulated that the fibronectin fibers may be cross-linked to the microfilament bundles via

tional 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 113

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specific membrane proteins (5, 27), but the evidence of this association is not conclusive. During the process of transformation with tumor viruses, fibroblasts become less adherent and less spread on the substrate. This is reflected in a decrease of both actin cables (7, 25, 29) and fibronectin fibers (21). These data implicate both the actin and the fibronectin fibers as having important roles in cell attachment and in the regulation of cell morphology. In this paper, we examine the processes of attachment and spreading in normal and Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts when the cells are trypsinized and then plated in the presence of cytochalasin B (CB). In vitro, CB prevents the assembly of actin into filaments (3, 8, 20). When cultured cells, after being spread on a substrate, are exposed to CB, the microfilament bundles are dramatically altered and are either fragmented (22, 24) or reorganized into discrete packets of microfilamentous material (14, 23, 30). We have shown previously that CB causes different morphological responses in normal and RSV-transformed chicken fibroblasts when added to cells already spread on a substrate (22). The nontransformed cells retract their cytoplasm, leaving behind long, dendritic-like processes (22); this CB response is characteristic of fibroblasts (6). The RSV-transformed cells round up but remain attached to the substrate (22). The roles of the intermediate filaments, the microfilament bundles, and the fibronectin matrix in the attachment and spreading of these cells after the cells are plated in the presence of CB are examined in this paper. Our data suggest that the intermediate filaments may play an important role in the determination of cell morphology in CB, particularly in the formation and maintenance of the pseudopodial-like processes in the nontransformed cells. The formation of these processes, a limited form of cell movement, occurs without the involvement of the microfilament bundles or the fibronectin matrix. The specific role of the intermediate filaments in the determination of cell morphology in CB, particularly in the formation of processes, remains to be determined. MATERIALS AND METHODS Cells. Dermal fibroblasts were prepared from 10- to 11-day-old chicken embryos (SPAFAS) and cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (Flow Laboratories, Inc.). Cells were infected with RSV tsLA24A at a multiplicity of infection of 1 to 5 focus-forming units per cell. The cells were 95% transformed within 2 to 3 days. After transformation, the cells were shifted to the nonpermissive temperature (41°C) and maintained for several passages. For experimental procedures these infected cells or normal uninfected cells were trypsinized and seeded at 2 x 105 cells per 35-mm dish and

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incubated at either 41 or 36°C. The cells were grown at this low density only for the duration of the treatments (1 to 3 days). Drug treatment. CB (Aldrich Chemical Co.) was added to the normal growth medium at 5 p.g/ml. Antisera. Anti-intermediate filament antiserum was obtained from a mouse hybridoma line producing antibodies to the fibroblast intermediate filament protein, vimentin, which is the major constituent of the intermediate filaments (S. Tapscott, unpublished data). A rabbit antibody with similar specificity for the vimentin intermediate filament protein has been described by Franke et al. (10). In this paper we call this antiserum anti-vimentin. Anti-actin was obtained and characterized by J. Schollmeyer. Anti-fibronectin was obtained from a rabbit injected with fibronectin purified from chicken embryo fibroblasts as described below. Immunofluorescence. Cells grown on glass cover slips were fixed with phosphate-buffered (pH 7.3) 3% formaldehyde for 7 min at 22°C, rinsed in phosphatebuffered saline, and fixed in acetone at -20°C for 10 min. The cover slips were incubated with antiserum for 30 to 60 min at 37°C, rinsed in several changes of phosphate-buffered saline, incubated with fluoresceinconjugated goat anti-rabbit immunoglobulin G (IgG) or rhodamine-conjugated goat anti-mouse IgM, mounted in Elvanol, and observed in a Zeiss microscope with epifluorescence. Electron microscopy. Cell monolayers were rinsed with 0.1 M sodium cacodylate buffer (pH 7.4) and fixed with 3% glutaraldehyde in cacodylate buffer for 30 min at room temperature. The cells were rinsed again and postfixed with 2% osmium tetroxide in cacodylate buffer for 15 min at room temperature. Monolayers then were stained with uranyl acetate (0.5% in water) for 1 h, dehydrated with a graded series of ethanol, infiltrated with an ethanol-Epon 812 mixture, and embedded in Epon 812. The tissue culture dish was pried away before sectioning. Thin sections (80 nm) were observed in a JEOL 100B electron microscope at 100 kV after being stained with uranyl acetate and lead citrate. Fibronectin purification. Fibronectin was extracted from chicken embryo cells, purified on a Sepharose CL-4B column, and dialyzed against serum-free medium immediately before use. Purified fibronectin at 50 ,ug/ml was capable of causing both the early attachment of cells to a tissue culture substrate and the increased spreading and flattening of transformed cells.

RESULTS Cell morphology after plating in the presence of CB. Fibroblasts, when exposed to trypsin as in routine cell subculture, retract their cytoplasm, round up, and detach from the substrate. After trypsinization, the membrane-associated microfilament bundles and peripheral microfilaments no longer can be detected and appear to be completely broken down (2, 11, 12). At areas of plasmalemmal blebbing, some condensed microfilaments are found (11). It has been shown in vitro that the presence of the drug CB inhibits the polymerization of actin into filaments (3, 8,

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20). In light of these facts, trypsinized cells were plated in the presence of CB to examine cell attachment, spreading, and movement under these conditions. CB was used at a concentration known to disrupt microfilament bundles when added to cells already spread on a substrate. To compare these cell functions in normal and transformed cells, these experiments were performed with chicken embryo dermal fibroblasts infected with tsLA24A, a temperature-sensitive transformation mutant of RSV. For controls, all experiments were performed in parallel with both uninfected cultures and cells infected with wild-type RSV Prague A. Figure 1 shows the time course for the spreading of tsLA24A-infected chicken embryo fibroblasts in the presence of CB at both the permissive (36°C) and the nonpermissive (41°C) temperatures for transformation. Cells grown at the permissive temperature quickly attached to the substrate, but remained round in CB. This parallels the results obtained when RSV-transformed cells, already spread on a tissue culture substrate, are exposed to CB (22). Figure la-c shows the absence of cell spreading at all times after plating in CB, even as late as 48 h in the presence of the drug. Short, blunt processes occasionally could be observed (Fig. lb, arrow). This occurred in only a few percent of the cells. Identical results were obtained with cells infected with the wild-type RSV. In contrast, cells incubated at the nonpermissive temperature for transformation (Fig. ld-f), like uninfected cultures, exhibited a considerable alteration of cell morphology when plated in CB. The morphological changes which occurred in CB involved extensive movement of the cytoplasm along the substrate in the formation of dendritic-like cellular processes. These processes were both extended and retracted in CB (determined by time lapse photography) (data not shown) and could be found in excess of 70 ,um in length and 5 pum in width. Routinely, the first morphological changes in the trypsinized cells plated in CB at the nonpermissive temperature were observed within a few hours after plating. These changes included a general flattenig of the cytoplasm around the nucleus in a symmetrical manner and the extension of very short pseudopodial processes from the central cell body. These processes usually were rather thick with blunt ends, but occasional cells with short, fine processes were observed. Figure ld exemplifies the morphological types present in early cell spreading in CB. Within 24 h after plating in CB, the majority of the cells had extended a number of pseudopodial processes (Fig. le). Although the size and number of processes varied from cell to cell, the extension of the processes by 24 h in CB was

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often quite remarkable (Fig. le, arrow). The formation of these processes in CB is of particular importance because it occurred under conditions which are known to disrupt the microfilament bundles. By 48 h in CB, the formation, extension, and branching of processes in these cells was very extensive (Fig. lf). All of the cells extended numerous processes of significant length (arborization), some extending more than 70 p.m from the central cell body. In the results presented here, arborization occurred by the extension and branching of dendritic-like processes from the central body of trypsinized cells plated on a tissue culture substrate in the presence of CB. This is the first description of arborization in which it is apparent that the processes do not form merely by the retraction of the cytoplasm on an already existing structure or structures, but rather that processes do form in CB as an active event involving considerable movement of the cell boundary as the processes are extended out along the substrate. In these experiments we have shown that the spreading of normal cells is altered when the cells are plated in CB and that the final morphology attained is similar to that which occurs when cells already spread on a substrate are exposed to CB (22). The following experiments describe the state of organization of the cytoskeleton in cells plated in the presence of CB. Localization of actin and the fibroblast intermediate filament protein, vimentin, in cells plated in the presence of CB. tsLA24A-transformed chicken fibroblasts were fixed and examined by double indirect immunofluorescence microscopy 24 to 48 h after being plated in the presence of CB. Antisera to both actin and vimentin were used to describe the relative states of assembly of the microfilaments and the intermediate filaments. The exposure of cultured cells to CB results in the disruption or reorganization of the microfilament bundles (14, 22, 24, 30). When trypsinized cells, either normal or transformed, were plated in the presence of CB, microfilament bundles did not form. This was demonstrated by indirect immunofluorescence staining with an actin antibody (Fig. 2b and d) and by electron microscopy studies (Fig. 3 and 4). No staining of microfilament bundles was detected in either the round or the arborized cell morphology. Of particular note is the apparent absence of microfilament staining in the processes of the arborized cells, especially when compared with the parallel staining of intermediate filaments with anti-vimentin (Fig. 2c). The actin staining revealed only short rods or spots of actin-containing structures. These may correspond either to short, condensed filamentous structures or to adhesion areas within the cell. Figure 2a and c demonstrates that intermedi-

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FIG. 1. Phase-contrast micrographs of tsLA24A-infected dermal fibroblasts plated at 2 x 105 cells per 35-mm petri dish in Eagle medium containing 10% fetal calf serum and 5 ,ug of CB per ml. Cells were grown either at 36'C, the permissive temperature for transformation (a-c) or at 41°C, the nonpermissive temperature (d-f). The micrographs were taken 6.5 h (a and d), 24 h (b and e), or 48 h (c and f) after plating in CB. The arrow in panel b denotes a short process seen occasionally early after plating in CB at 36°C. The arrow in panel e denotes a particularly long process seen at 41°C, only 24 h after plating in CB. Bar, 20 p.m.

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FIG. 2. Double indirect immunofluorescence of tsLA24A-infected dermal fibroblasts 48 h after being plated at 2 x 105 cells per 35-mm petri dish in Eagle medium containing 10%o fetal calf serum and 5 ,ug of CB per ml. (a and b) cells grown at 36°C, same field (c and d) cells grown at 41°C, same field. (a and c) Immunofluorescence with a monoclonal antibody to vimentin; the secondary antibody was rhodamine-conjugated goat anti-mouse IgM. (b and d) Immunofluorescence with a rabbit antibody to actin; the secondary antibody was fluoresceinconjugated goat anti-rabbit IgG. Bar, 20 ,um.

ate filaments were present in RSV-infected cells grown in CB at both the permissive and the nonpermissive temperatures for transformation. However, the distribution of intermediate filaments was different in the round and the arborized cell morphologies. At the permissive temperature, the filaments were randomly oriented throughout the cytoplasm of the round cells. Often, due to the roundness of these cells individual filaments could not be discerned. This resulted in a diffuse fluorescence image. At the nonpermissive temperature, the intermediate filaments were found concentrated in the processes. The filaments of the central cell body seem to be directed toward the dendritic-like processes. Therefore, it appears that the organization of

intermediate filaments in cells plated in the presence of CB is altered by transformation with RSV. Electron microscopy of cells plated in the presence of CB. Dermal fibroblasts infected with tsLA24A as described above were plated on

collagen-coated dishes in the presence of CB, incubated for 48 h at either the permissive or the nonpermissive temperature for transformation, fixed, embedded, and sectioned for electron microscopy. Cells grown in CB at the permissive (Fig. 3) and nonpermissive (Fig. 4) temperatures for transformation exhibited their characteristic round and arborized morphologies, respectively (see Fig. 1). The electron micrographs revealed that cells at both temperatures exhibited amor-

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FIG. 3. Electron micrographs of a thin section of tsLA24A-infected dermal fibroblasts prepared for electron microscopic observation 48 h after being plated in the presence of CB at 36°C, the permissive temperature for transformation. (a) Low magnification (x8,100). (b) High magnification (x23,550) of area outlined in panel a. Note the large amorphous region (arrow).

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FIG. 5. Immunofluorescence with rabbit antiserum to fibronectin purified from chicken embryo fibroblasts. Uninfected chicken embryo dermal fibroblasts were plated at 2 x 105 cells per 35-mm petri dish in Eagle medium containing 10% fetal calf serum, allowed to spread for 24 h at 37°C, and then exposed to CB for 15 min (a) or 24 h (b) before fixation for immunofluorescence. (c) Uninfected chicken embryo dermal fibroblasts plated at 2 x 105 cells per 35-mm petri dish in Eagle medium containing 10% fetal calf serum and 5 ,ug of CB per ml and grown for 24 h in the presence of the drug before fixation for immunofluorescence. Bar, 20 ,m.

phous regions which were somewhat granular in appearance and did not contain cell organelles. These regions were most prominant in the transformed cells (Fig. 3, arrows). Microfilament bundles were not found at either temperature (Fig. 3 and 4). In fact, it was difficult to ascertain the presence of any microfilament fibers. Intermediate filaments were found in cells grown at either temperature but were predominant in the dendritic processes of the arborized cells grown at the nonpermissive temperature, where they were often found in highly organized and concentrated parallel arrays (Fig. 4). These filaments were also found concentrated in the central cell body of the nontransformed cells plated in CB, where they usually appeared to be directed towards the processes (Fig. 4a, arrow). At the permissive temperature for transformation, cells plated in CB contained only sparse and randomly oriented intermediate filaments in thin section. Only occasional microtubules were

identified in the dendritic processes of the arborized cells. The low concentration of microtubules in these processes makes it unlikely that they are functioning in a structural capacity. Role of fibronectin in CB-treated cells. Since the fibronectin matrix has been implicated as a major component in the attachment and spreading of cells in culture, its role in the maintenance of cell morphology in CB was examined. Uninfected dermal fibroblasts were plated and allowed to attach and spread on a tissue culture substrate for 24 h. The medium then was removed and replaced with fresh medium containing CB. At 15 min after the addition of CB, which was sufficient time for the disruption of the microfilament network and after arborization of these cells had begun, we still were able to demonstrate the maintenance of a complete, although perhaps somewhat distorted, fibronectin matrix by indirect immunofluorescence (Fig. Sa).

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FIG. 6. tsLA24A-infected chicken embryo dermal fibroblasts plated at 2 x 105 cells per 35-mm petri dish in Eagle medium containing 10% fetal calf serum and grown at 41°C (a) or 36°C (b) for 24 h before fixation for immunofluorescence staining with rabbit antiserum to fibronectin. Note the loss of the fibronectin matrix at the permissive temperature. Bar, 20 ,um.

By 24 h after the addition of CB, the filamentous network of fibronectin had been largely lost. Only nonfilamentous regions associated with the cell body and a few short fibers associated with the surrounding substrate were revealed by immunofluorescence studies with the fibronectin antibody (Fig. 5b). In particular, the cell processes showed little fibronectin. The release of fibronectin protein in the presence of CB has been examined biochemically by Ali and Hynes (1). Our data indicate that the disruption of the microfilament network by CB precedes and may lead to the disruption of the fibronectin network, although one is not immediately dependent upon the other. The loss of the fibronectin matrix after exposure to CB occurs in the absence of translational movement, which is inhibited by CB (4, 31). In the next experiment, uninfected dermal fibroblasts were plated directly into CB, and the cells were fixed after 24 h in the presence of the drug, which is sufficient time for arborization to occur. Indirect immunofluorescence with fibronectin antibody demonstrated a diffuse staining pattern on the central cell body (Fig. 5c) which was similar to that seen in cells treated for 24 h with CB after they had spread on a substrate (Fig. 5b). However, no assembled fibronectin matrix was observed on the substrate, and the cell processes again were particularly devoid of fibronectin. Fibronectin involvement in the determination of cell morphology of RSV-infected fibroblasts plated in CB. When dermal fibroblasts infected with tsLA24A had spread in the absence of CB, a network of fibronectin filaments was estab-

lished on the substrate at both the permissive and the nonpermissive temperatures for transformation (Fig. 6). However, a more extensive fibrous network, similar to that of uninfected cells, was present at the nonpermissive temperature (Fig. 6a). These cells were plated in the presence of CB at both the permissive and the nonpermissive temperatures for transformation and grown for 24 h in the presence of the drug. The resulting round and arborized cells were fixed and examined by double indirect immunofluorescence microscopy with antibodies to both vimentin and fibronectin to enable comparison of both the intermediate filaments and the fibronectin matrix of the same cell. In Fig. 7 are shown the results at the permissive (panels a and b) and the nonpermissive (panels c and d) temperatures for transformation with antibodies to vimentin (panels a and c) and fibronectin (panels b and d). Prominent intermediate filaments were observed, particularly in the dendritic-like processes at the nonpermissive temperature, as described above. However, there was very little staining of fibronectin at either temperature. More notable was the virtual absence of any fibronectin matrix. Similar results were obtained at both temperatures when tsLA24A-infected cells already spread on the substrate were exposed to CB. The results at the nonpermissive temperature were similar to those obtained with uninfected cultures. However, the infected cells plated and grown in CB at the nonpermissive temperature had even less fibronectin associated with them (Fig. 7b) than did similarly treated uninfected

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FIG. 7. Double indirect immunofluorescence of tsLA24A-infected dermal fibroblasts 48 h after being plated at 2 x 105 cells per 35-mm petri dish in Eagle medium containing 10% fetal calf serum and 5 ,ug of CB per ml. (a and b) cells grown at 36°C, same field (c and d) cells grown at 41°C, same field. (a and c) Immunofluorescence with a monoclonal antibody to vimentin; the secondary antibody was rhodamine-conjugated goat anti-mouse IgM. (b and d) Immunofluorescence with a rabbit antibody to fibronectin; the secondary antibody was fluorescein-conjugated goat anti-rabbit IgG. Bar, 20 p.m.

cells (Fig. Sc). The results with both infected and uninfected cultures suggest that the fibronectin matrix does not play a role in process formation in CB. These results also indicate that the loss of fibronectin in transformed cells is probably not responsible for the inability of these cells to form processes in CB. Experiments performed with exogenously added fibronectin substantiate this conclusion. Fibronectin was added at a concentration of 100 ,ug/ml to RSV-infected cells grown at the permissive temperature for transformation before the cells were exposed to CB. The presence of exogenously added fibronectin did not change the morphological response of transformed cells to CB. Also, the addition of fibro-

nectin (100 ,ug/ml) to infected cultures at the permissive temperature which were already in the presence of CB did not cause any change in their morphology; they remained round. Therefore, although fibronectin is capable of causing a morphological reversion of transformed cells via the flattening of their cytoplasm, exogenously added fibronectin failed to induce the formation of processes in transformed cells exposed to CB. When these experiments were carried out at the nonpermissive temperature for transformation, the addition of fibronectin resulted in the increased adhesion of the cytoplasm in the pseudopodial processes and consequently in the flattening of the processes of the arborized cells. From these results we conclude that the differ-

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ences in the fibronectin matrix between normal and RSV-transformed cells are not responsible for the differential response of normal and transformed cells to CB. DISCUSSION Different morphological response of normal and transformed cells plated in CB and the role of microfilaments in this process. The spreading of trypsinized cells on a tissue culture substrate requires a number of cellular functions, particularly those involving the structural proteins and, primarily, the microfilaments (12, 18, 19, 28). In spread cells the microfilament bundles are fragmented (22, 24) or reorganized (14, 23, 30) after exposure to CB, and the cells become arborized, a morphology characterized by the retraction of the cytoplasm to the central body of the cell, leaving dendritic-like processes behind (6, 16). We found that this arborized morphology also was attained when trypsinized cells, which are round and deficient in microfilament bundles, were plated on a tissue culture substrate in the presence of CB. CB inhibits the formation of actin filaments in vitro (3, 8, 20). In our experiments, CB appeared to inhibit the formation of microfilament bundles in vivo. Movement of the cytoplasm out along the substrate occurred slowly when cells were plated in the presence of CB, but within 24 h the round cells had extended long branched processes which characterize the arborized cell morphology. The formation of these processes involves a considerable extension of the cell boundary and therefore a type of cell movement. Our data indicate that the extension of these structures occurs in the absence of microfilament bundles. These results represent the first demonstrations of process formation in CB as both an active function of the cell and a type of cell movement which is not dependent upon the microfilament bundles. Cells transformed with RSV which were trypsinized and plated in the presence of CB attached to the substrate but remained round. We have demonstrated this altered response previously with transformed cells which have been treated with CB after spreading on a substrate, and we have shown that the expression of the transforming protein pp6Osrc prevents either the establishment or the maintenance of processes in the presence of CB (22). This indicates that there is an as yet unidentified structural alteration in the transformed cells. Organization of fibronectin in cells plated in the presence of CB. The attachment of cells in CB, and the formation of processes by these cells, is not mediated by an organized fibronectin matrix. Furthermore, the correlation between a disruption of the microfilament network of spread fibroblasts by CB and the subsequent,

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although not immediate, loss of the fibronectin network suggests that the maintenance and possible establishment of the fibronectin matrix may be dependent on the presence of the microfilament bundles. This hypothesis is supported by studies which show the colinearity of these two structural networks (15, 17, 26) and suggest linkage through the membrane via specific membrane proteins (5, 27). Although the loss of substrate adhesion in transformed cells has been attributed both to a loss of fibronectin matrix (21) and to an alteration of microfilament bundles (7, 25, 29), neither of these transformationrelated changes accounts for the difference in morphology of normal and RSV-transformed cells in CB. Altered cell-substrate adhesion produced by CB. Whether the extension of processes involves the pushing action of the intermediate filaments or a pulling action due to altered substrate adhesion is difficult to determine. However, several additional observations may be relevant in this context. Cells plated directly into CB-containing medium adhered more rapidly to the substrate than cells plated in normal medium. This was also true for tsLA24A-infected cells incubated at either the permissive or the nonpermissive temperature for transformation. Since cells plated at the permissive temperature remained round in CB, the altered adherence which was observed in cells plated in the presence of CB at both temperatures probably does not play a role in the ability of the cells at the nonpermissive temperature to extend processes. Also, in preliminary experiments, fibroblasts plated on plastic bacterial culture dishes, which normally do not allow cell spreading, still allowed the extension of the dendrite-like processes in CB. Since a substrate which does not permit normal cell spreading will still allow cells to aborize in CB, the usual cell-substrate interactions involved in cell spreading may not be required for process formation in CB. Structural elements involved in process formation in CB. The results presented here demonstrate that the extension and maintenance of processes in CB are not dependent upon either the microfilament or the fibronectin matrixes. However, these filament structures are implicated in both the establishment and the maintenance of the normal morphology of fibroblasts. Although translational movement of cells is inhibited by CB, the extension of processes in CB demands considerable cytoplasmic movement. An ultrastructural analysis of nontransformed cells plated in CB raises the possibility that intermediate filaments may play a structural as well as a motile role in the formation or maintenance of the pseudopodial process characteristic of these cells. The intermediate filaments pre-

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dominated as highly organized, tightly packed parallel arrays in these processes. The intermediate filaments in RSV-transformed cells, which remain round after plating in CB, were highly disorganized and found scattered throughout the round cell body. This implies a structural alteration of the intermediate filaments in the transformed cell, an alteration which is dependent on the expression of pp6osrc (see 22). It is important to note that process formation in CB may also require the structural elements involved in cellsubstrate adhesion and that these structures are probably altered by transformation with RSV as well. Cells plated in the presence of CB also contained amorphous regions which were somewhat granular in appearance. They were found throughout the main body and the processes of the nontransformed cells, but were most prominent in the round transformed cells (Fig. 3). Although these regions may correspond to the areas of condensed or reorganized microfilamentous material described previously for cells exposed to CB (14, 23, 30), they are different in appearance. They are not as dense, nor have they been found to contain microfilaments (14, 30) or trap ribosomes (30) as has been described for the condensed microfilamentous material. These differences may stem from the fact that the cells in this study were plated in the presence of CB after trypsinization had produced round, microfilament-deficient cells, rather than having been exposed to CB after the establishment of their cytoskeleton was complete. It also has been demonstrated that intermediate filament protein can exist as nonfilamentous structures in mitotic cells (9). Since the round transformed cells plated in CB lacked the high concentration of intermediate filaments present in similarly treated nontransformed cells, and since large areas of their cytoplasm were composed of the amorphous material, one can speculate that these amorphous regions can consist at least in part of intermediate filament protein. Experiments are now being pursued which address this question. Through the use of CBtreated cells, in which both the microfilament and the fibronectin matrixes have been disassembled, it may be possible to identify a positive role for the function of the intermediate filaments, to identify more clearly the elements of the cell involved in adhesion, and to understand how cell adhesion and structure are altered by transformation with RSV.

MOL. CELL. BIOL. the recipient of a Young Investigators Award from the National Institutes of Health. D.B. is a Leukemia Society of America Scholar. LITERATURE CITED 1. Ali, I. U., and R. 0. Hynes. 1977. Effects of cytochalasin B and colchicine on attachment of a major surface protein of fibroblasts. Biochim. Biophys. Acta 471:16-21. 2. Britch, M., and T. D. Allen. 1980. The modulation of cellular contractility and adhesion by trypsin and EGTA. Exp. Cell Res. 125:221-231. 3. Brown, S., and J. Spudich. 1981. Mechanism of action of cytochalasin: evidence that it binds to actin filament ends. J. Cell Biol. 88:487-491. 4. Carter, S. B. 1967. Effect of cytochalasins on mammalian cells. Nature (London) 213:261-264. 5. Chen, W. T., and S. J. Singer. 1980. Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata. Proc. Natl. Acad. Sci. U.S.A. 77:7318-7322. 6. Croop, J., and H. Holtzer. 1975. Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. J. Cell Biol. 65:271-285. 7. Edelman, G. M., and I. Yahara. 1976. Temperaturesensitive changes in surface modulating assemblies of fibroblasts by mutants of Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 73:2047-2051. 8. Flanagan, M. D., and S. Lin. 1980. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J. Biol. Chem. 255:835-838. 9. Franke, W. W., E. Schmid, C. Grund, and B. Geiger. 1982. Intermediate filament proteins in nonfilamentous 10.

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19. ACKNOWLEDGMENTS This work was supported by grants from the National Cancer Institute, the National Institute of General Medical Sciences, and the Muscular Dystrophy Association. A.S.M. is

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