Cytoskeletal organization, vinculin-phosphorylation, and fibronectin expression in transformed fibroblasts with different cell morphologies

VIROLOGY

151,50-65

(1986)

Cytoskeletal Organization, Vinculin-Phosphorylation, and Fibronectin Expression in Transformed Fibroblasts with Different Cell Morphologies ERICH

A. NIGG,*

BARTHOLOMEW M. SEFTON,? AND PETER K. VOGTQ1

S. J. SINGER,*

*In&it& fiir Zellbiologie. Eidgenksische Technische Hochxhule, Hkggerberg, CH-8093 ZG-ich, Switzerland; ~iWoLecnlar Biology and Virology Laboratory, The Salk Institute, P.0. Box 85800, San Diego, Califonia 92138; $Depcwtment of Biology, University of Califor?zia at San Diego, Lu Jolla, California 9.2033; and §Departnwnt of Microbiobg~, University of Southern California, S&o01 of Medicine, 2011 Zonal Avenue, Los Angeles, California 90033 Received September 19, 1985; accepted January

16, 1986

Neoplastic transformation of fibroblasts results in widely different cell morphologies. We have attempted to correlate cell morphology with cytoskeletal organization and Iibronectin expression in murine and avian fibroblasts transformed by a diverse group of viral and chemical agents. The distribution of vinculin, a-actinin, actin, and surface fibronectin was studied, and, where appropriate, also the extent of phosphotyrosine modification of vinculin. Irrespective of the transforming agent we found that increased cell roundingwas generally correlated with a reduction in vinculin-containing focal adhesions, a dissolution of microfilament bundles, and a reduction of extracellular fibroneetin. In contrast, spindle-shaped fibroblasts expressed relatively high levels of surface fibronectin. Reorganization of vinculin, actin, and a-actinin into rosette-like structures was observed in polygonal or rounded cells transformed by viruses encoding tyrosine kinases, but was not seen in fibroblasts transformed by agents without associated tyrosine kinase activity or in spindle-shaped cells. No correlation was found between the extent of phosphotyrosine modification of vinculin and the extent of cell rounding. Irrespective of cell morphology, the extent of tyrosine phosphorylation of vinculin was high in all cells transformed by viruses carrying the src gene, but low in those transformed by viruses expressing the fps gene. Our results indicate that the morphology of a transformed cell is determined by a combination of several factors which are affected to different extents by different transforming agents. 0 1986 Academic Press, Inc. INTRODUCTION

Progress has been made in defining the structures and molecular components involved in determining the shape and adhesion sites of normal flbroblasts (Geiger et ah, 1980; Chen and Singer, 1980, 1982; Geiger 1983). In the present studv attention has-been focused upon three “structural comnonents which contribute to the morphofogy of normal cells, namely, focal adhesion plaques, microfilament bundles, and the extracellular matrix. In wellspread normal fibroblasts, focal adhesion plaques are pun&ate areas of closest approach of the ventral cell surface to the substrate. They are the sites of strongest adhesion (Abercrombie and Dunn, 1975; Harris, 1973). Inside the cell at the sites of the focal adhesions, bundles of actin-con-

Changes in cell shape and cell adhesion accompany neoplastic transformation of fibroblasts. Normal fibroblasts in culture are flat and adhere firmly to their substrate. After oncogenic transformation, fibroblasts change their shape to different extents, depending on the transforming agent. Transformed fibroblasts can assume flat, spindle-shaped, polygonal, or rounded morphologies. They are also less adherent than their normal counterparts. The molecular and structural bases for these changes in cell shape and adhesion are still poorly understood. ‘To whom dressed.

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50

FIBRONECTIN

IN TRANSFORMED

taining microfilaments terminate (Abercrombie et al, 1971; Heath and Dunn, 1978), and the proteins vinculin and a-actinin are concentrated (Geiger, 1979; Chen and Singer, 1982). Starting from the focal adhesions, microfilament bundles extend through the cytoplasm and are often closely associated with the ventral or dorsal surfaces of the cell (Abercrombie et al., 1971; Abercrombie and Dunn, 1975), functioning as part of the cytoskeleton and contributing to the flattened shape of the cell. On the outer surfaces of the cell, a set of components including the protein fibronectin forms a reticular network (Hynes, 1976; Vaheri et al, 1978). This extracellular matrix imparts rigor to the cell shape and contributes to adhesiveness to the substrate. There have been several studies of cultured transformed cells with different morphologies (e.g., Royer-Pokora et al., 1978; Anderson et ab, 1981; Carley et al., 1981; Rohrschneider et aZ., 1981; Fujita et ak, 1981; Guyden and Martin, 1982; Iwashita et al., 1983; Rohrschneider and Rosok, 1983; Notter and Balduzzi, 1984; Di Renzo et ah, 1985; Tarone et al, 1985). The changes in cell shape that accompany transformation may result from alterations in either the abundance or function of the proteins which comprise the cytoskeleton and the extracellular matrix. There is reason to think that the ability of vinculin to contribute to cell shape and adhesion may be interfered with by a number of related viral transforming proteins. This polypeptide component of the focal adhesion plaques is modified by phosphorylation at tyrosine by the transforming proteins of RSV, Y73 avian sarcoma virus and Abelson murine leukemia virus (Sefton et ab, 1981). The morphologies of transformed cells are not uniform. To examine the molecular basis of the transformed phenotype, we have characterized the organization of the cytoskeleton and the extracellular matrix of cells transformed by a diverse group of agents. Because the adhesion plaques in cells transformed by RSV undergo an unusual reorganization, particular attention has been paid to the structure of those adhesion plaques that persist in trans-

51

FIBROBLASTS

formed cells. The distributions of intracellular vinculin, a-actinin and actin-containing microfilaments, as well as the expression of surface fibronectin, were examined by immunofluorescence microscopy. Microscopic observations on the deposition of fibronectin in the extracellular matrix have in some cases also been corroborated by enzymatic iodination of the transformed cultures. In addition, it was of interest to investigate the possible correlation of cell shape with the level of phosphotyrosine modification in vinculin. We therefore have measured the extent to which vinculin is phosphorylated on tyrosine in cells infected with retroviruses that encode tyrosine-specific protein kinases. MATERIALS

AND

METHODS

Cells and cell culture. The cultivation of NIH Swiss 3T3 cells (Todaro and Green, 1963) and their derivatives transformed by et ab, benzo[u]pyrene-BP3T3 (Holley 1976), SV40 virus-SV3T3 (Todaro et ak, 1964), and the P120 strain of Abelson murine leukemia virus (A-MLV)-ANN-1 (Scher and Siegler, 1975) has been described previously (Sefton et aZ., 1980). Similarly, the cultivation of BALB/3T3/ A31 cells (Aaronson and Todaro, 1968) and their derivatives transformed by SchmidtRuppin Rous sarcoma virus, subgroup D (SR-RSV-D)-SR3T3 and Kirsten murine sarcoma virus (KiSV) (Aaronson and Weaver, 1971) has been described (Sefton et al, 1980). The Moloney murine sarcoma virus (MoMSV)-transformed mouse cells were from a CFW/D mouse (Ball et aZ., 1973) and were grown as described (Sefton et al, 1980). Chick cells infected with the avian retroviruses PRCII, PRCIIp, PRCIV (Breitman et aZ., 1981a, b), Y73 (Kawai et aL, 1980), Fujinami sarcoma virus (FSV), MC29, SR-RSV, subgroups A and D (Duff and Vogt, 1969), the fusiform Fl variant of SR-RSV (Martin, 1971) or the fusiform variant of the Bryan high titer strain of RSV (ffRSV) (Yoshii and Vogt, 1970), were prepared on plastic petri dishes as described before (Sefton et d, 1978) and were reseeded on glass coverslips in medium containing 1% calf serum after they had

52

NIGG

undergone morphological transformation. The QT-6 line of methylcholanthrenetransformed quail cells (Moscovici et al., 1977) was cultivated in the same manner as the virally transformed chicken cells. Immunochemical reagents. The preparation and affinity-purification of guinea pig antibodies to chicken gizzard vinculin and rabbit antibodies to chicken gizzard cw-actinin, as well as the preparation of rhodamine- and fluorescein-conjugated goat antibodies to rabbit IgG and guinea pig IgG were described previously (Geiger, 1979). To stain fibronectin on chick fibroblasts, affinity-purified guinea pig antibodies to chicken serum fibronectin were used, while for mouse cell fibronectin, rabbit antibodies raised against human serum fibronectin were employed (Little and Chen, 1982). F-actin was stained with 7nitrobenz-2-oxa-1,3 diazole phallacidin (NBD-phallacidin) purchased from Molecular Probes, Inc. (Junction City, Oreg.). Immunoj7Tuvrescence microscopy. Cells were cultured on 18-mm2 glass coverslips for the times indicated in the figure legends, in all cases for at least 40 hr before immunolabeling experiments. For staining of intracellular proteins, cells were fixed for 5 min at room temperature with 3% formaldehyde/2% sucrose in phosphatebuffered saline (PBS), pH 7.6, washed 3 times in PBS and subsequently permeabilized by treatment for 5 min with 0.5% Triton X-100 in 10 mM HEPES buffer containing 200 mM sucrose, 3 mM MgC12, 50 mMNaC1, pH 7.4. For double indirect immunofluorescence labeling, the two primary antibodies were mixed, and the two secondary reagents were mixed before being applied to the cells. NBD-phallicidin was added to the cells simultaneously with rhodamine-conjugated secondary antibodies. Incubations with antibody reagents were for 10 min at room temperature. For immunofluorescent labeling of extracellular fibronectin, cells were fixed as described above but were not permeabilized by detergent treatment. Affinity-purified antibodies to vinculin and a-actinin were used at 10 pg/ml for labeling chicken fibroblasts and at 40-50 pg/ml for mouse fibroblasts. Affinity-purified antibodies to chicken fi-

ET AL.

bronectin were used at lo-15 pg/ml, while mouse fibroblasts were labeled with affinity-purified antibodies to human fibronectin at 15 @g/ml or, in most cases, with an antiserum to human fibronectin diluted l/200. In the last two cases, the results were indistinguishable. Epifluorescence observations were made with a Zeiss Photoscope III instrument. Phosphoaminoacid analyses. Confluent cultures of virally transformed or nontransformed chicken fibroblasts were incubated in lo-cm culture dishes and labeled with [32P]orthophosphate (New England Nuclear, carrier-free, 1 mCi/ml) for approximately 18 hr in 5 ml of phosphatefree medium. The labeled monolayers were washed once with PBS, and the cells were then dissolved in 1 ml of phosphate-RIPA buffer (1% Triton X-100,1 % sodium deoxycholate, 0.1% SDS, 0.15 MNaCl, 0.02 M sodium phosphate, pH 7.2) in the presence of 0.2 mM phenylmethylsulfonyl fluoride. Following eentrifugation of the extracts 100,000g for 30 min, the supernatants were removed and the vinculin was immunoprecipitated from them and analyzed for phosphoaminoacids as described (Sefton et al., 1981). Parallel cultures were used for determination of total cellular phosphotyrosine in order to ascertain comparable extents of transformation by different viruses. Lactoperoxidase-catalyzed

iodination.

Cells to be labeled by iodination were subcultured to 35-mm plastic dishes and allowed to grow at least 72 hr prior to labeling. The cell monolayer was then washed twice with phosphate-buffered saline. Iodination was carried out in 1 ml of phosphate-buffered saline containing 5 mM glucose and 300 &i ‘251-sodium (New England Nuclear; carrier-free). Labeling was initiated by the addition of 0.625 units of lactoperoxidase (Calbiochem) and 0.125 units of glucose oxidase (Calbiochem) and allowed to proceed for 15 min at room temperature. The labeled cells were washed twice with phosphate-buffered saline and then dissolved in SDS-polyacrylamide gel sample buffer (2% SDS, 0.1 M dithiothreitol, 5% 2-mercaptoethanol, 5 mM sodium phosphate, pH 7.0,10% glycerol). To ensure

FIBRONECTIN

IN TRANSFORMED

quantitative solubilization of fibronectin, the dish was scraped vigorously with a rubber policeman. Labeling of fibronectin was analyzed by SDS-polyacrylamide gel electrophoresis and quantified by excision of the labeled protein and determination of incorporated iodine using a gamma counter. To ensure that equal quantities of cellular protein were analyzed, all analytical gels were stained for protein. RESULTS

Morphology

of Transfied

ST3 Cells

In Fig. 1 the structural characteristics of several transformed mouse fibroblast cell lines (Figs. lE-T) are compared to those of normal BALB 3T3 cells (Figs. lAD). Transforming agents were benzo[a]pyrene (Figs. lE-H), Moloney murine sarcoma virus (MoSV), which carries the mos oncogene, (Figs. 11-L), Schmidt-Ruppin RSV of subgroup D (SR-RSV-D), which carries the src oncogene, (Figs. lM-P) and Abelson murine leukemia virus, which carries the abl oncogene (Figs. lQ-T). Although not shown pictorially here, SV40transformed NIH-3T3 cells showed characteristics very similar to those of the benzpyrene-transformed 3T3 cells, and cells transformed by Kirsten murine sarcoma virus (KiSV), which carries the ras oncogene, resembled the MoSV-transformed cells (Table 2). Transformation of mouse fibroblasts is accompanied by different degrees of cell rounding, depending on the transforming agent (Fig. lD, H, L, P, T). The benzpyrenetransformed cells had a morphology very similar to that of 3T3 cells. In contrast, the RSV-transformed cells were highly rounded. Several parameters were found to correlate with the transition from flat to round cell shape. First, immunolabeling of vinculin in focal adhesion plaques (Fig. lA, E, I, M, Q) showed progressive reduction with increased rounding. As observed previously (David-Pfeuty and Singer, 1980; Nigg et al., 1982), vinculin in RSV-transformed cells was largely reorganized into patches of rosette-like clusters often underlying the nucleus near the ventral cell surface (Fig. 1M). Occasionally similar

53

FIBROBLASTS

vinculin rosettes were observed in Abelson virus-transformed fibroblasts (not shown; see also Rohrschneider and Najita, 1984). We did not observe vinculin rosettes in any other mouse cell lines examined here. A&in-containing microfilament bundles (Fig. lB, F, J, N, R) became less prominent in proportion to the decreased labeling of vinculin in focal adhesion plaques. In RSV- and Abelson virus-transformed cells, where vinculin was reorganized into rosette structures, actin was found invariably to codistribute with vinculin (Fig. 1N). A progressive decrease in extracellular fibronectin also correlated with increased cell rounding (Fig. lC, G, K, 0, S). Morphology

of Transfmd

Chick Cells

Figure 2 shows the results of a similar analysis carried out on chick embryo fibroblasts (Figs. 2A-D). The transforming viruses were SR-RSV-A (Figs. 2E-H), ffRSV, a variant of RSV which induces a spindleshaped cell morphology (Figs. 21-L), and avian sarcoma viruses PRCIV (Figs. 2MP) and PRCII (Figs. 2Q-T), both of which carry the fps oncogene. Although these four viruses all encode tyrosine protein kinases, they produce different transformed cell morphologies. Wild-type RSV caused extensive cell rounding (Fig. 2H), and a redistribution of vinculin (Fig. 2E) and actin (Fig. 2F) into rosette structures. The fusiform variant of RSV induced a spindle-shaped cell morphology (Fig. 2L). These cells contained a reduced number of focal adhesion plaques which were present only at the poles of the elongated cells (Fig. 21); we did not observe any redistribution of vinculin into rosettelike structures in these cells. The number of actin cables was reduced, but microfilaments were not completely absent (Fig. W). Very similar results were obtained with chick embryo fibroblasts transformed by Fl (Martin, 19’71), another fusiform variant of RSV (not shown). The avian sarcoma virus PRCII also induced spindle-shaped morphology in transformed cells (Fig. 2T). The distributions of vinculin (Fig. 2Q) and actin (Fig. 2R) in these cells were very similar to those

FIG. 1. (A) Normal and transformed mouse fibroblasts grown for 2 days to about 60% confluence. A given culture was doubly labeled by immunofluorescence for vinculin (panels A, E, I, M, Q) and actin (panels B, F, J, N, R, respectively). Pairs A, B: normal BALB 3T3; E, F: benzpyrene-transformed NIH 3TS; I, J: MoMSV-transformed mouse cells; M, N: SR-RSV-transformed BALB 3T3; Q, R: AbMuLV-transformed NIH 3T3. Arrowheads in M and N point to a condensed cluster of vinculin and actin patches (a “rosette”), while the arrows in M and N designate a region of spread-out

patches. All panels at same magnification; bar in Q indicates 20 pm. (B) Normal and transformed mouse cells grown for 3 days to about 80% confluence. A given culture was examined by Nomarski optics (panels D, H, L, P, T) and fluoreseently labeled for fibronectin (panels C, G, K, 0, S, respectively). Pairs C, D: normal BALB 3T3; G, H: benzpyrene-transformed NIH 3T3; K, L: MoMSV-transformed cells; 0, P: SR-RSV-transformed BALB 3T3; S, T: AbMuLV-transformed NIH 3T3. All panels at same magnification; bar in S indicates 20 pm.

FIG. 2. (A) Normal and transformed chick embryo fibroblasts (CEF) grown for 2 days to about 50% confluence. A given culture was doubly labeled by immunofluorescence for vinculin (panels A, E, I, M, Q) and aetin (panels B, F, J, N, R, respectively). Pairs A, B: normal CEF; E, F: RSVtransformed CEF, I, J: CEF transformed by ff-mutant of RSV; M, N: PRC-IV-transformed CEF, Q, R: PRC-II-transformed CEF. Arrows in E, F, and Q, R designate residual focal adhesions in the transformed cells. All panels at same magnification; bar in Q indicates 20 pm. (B) Normal and

transformed chick embryo fibroblasts (CEF), grown for 2. days (C, D, 0, P; and S, T) or 3 days (G, H; K, L) to about 8040% confluence. A given culture was examined by Nomarski optics (panels D, H, L, P, T) and labeled by immunofluorescence for fibronectin (panels C, G, K, 0, S, respectively). Pairs C, D: normal CEF, G, H: RSV-transformed CEF; K, L: CEF transformed by ff-mutant of RSV, 0, P: PRC-IV-transformed CEF; S, T: PRC-II-transformed CEF. All panels at same magnification; bar in S indicates 20 pm.

NIGG

58

ET AL.

seen in ffRSV-transformed cells; again, we did not observe any redistribution of microfilament proteins into rosette-like structures (Figs. 2Q, R). In contrast, PRCIV virus caused cells to assume a morphology which we shall refer to as polygonal (Fig. 2P). Vinculin-containing focal adhesions were limited to a few sites at the cell periphery in these cells, and, occasionally, some redistribution of vinculin into rosette-like patches was seen (Fig. 2M). In both the polygonal PRCIV virus-transformed cells and the spindle-shaped PRCII virus-transformed cells, the distribution of actin was congruent with that of vinculin (Figs. 2N, R).

bryo fibroblasts transformed by RSV (Fig. 3C), Y73 virus, which carries the yes oncogene (Fig. 3E) or PRCIIp virus, which carries the fps oncogene (Fig. 3G), vinculin was redistributed into rosette-like structures with different degrees of compactness. We never saw such clusters in cells transformed by agents not affecting the phosphorylation of proteins on tyrosine, such as the avian myelocytomatosis virus MC29, which carries the myc oncogene (Fig. 31), or the chemical carcinogen methylcholanthrene (not shown, see Table 2). As has been shown for actin in Figs. 1 and 2, a-actinin also consistently redistributed with vinculin (Figs. 3B, D, F, H, J; see also David-Pfeuty and Singer, 1980).

Abundance of Fibrwectin in Cultures of Transformed Chick Cells

Phosphorylation of Vinculin on Tyrosine

As determined by immunofluorescence microscopy, both the highly rounded cells transformed by RSV and the polygonal cells transformed by PRCIV virus contained little surface fibronectin (Figs. 2G, 0). In contrast, the spindle-shaped cells resulting from transformation by either fusiform variants of RSV or PRCII virus exhibited an extensive network of extracellular fibronectin (Figs. 2K, S). These results were corroborated by measuring the amount of cell surface-fibronectin by lactoperoxidase-catalyzed iodination. Cultures of wild-type RSV-transformed cells expressed only about 25% of the surface fibronectin found in untransformed cells. In contrast, cells transformed by the Fl fusiform variant of RSV or by PRCII expressed 50 and 95% ,respectively, of the extracellular fibronectin of untransformed cultures. Vine&in-Containing

Rosettes

In some of the transformed cells with rounded or polygonal morphologies, vinculin was redistributed into rosette-like structures (See Figs. 1 and 2). These structures are illustrated more extensively in Fig. 3 in which the distributions of vinculin and cy-actinin are compared. In chick em-

Vinculin is phosphorylated on tyrosine to a significant extent in cells transformed by RSV (Sefton et al, 1981). It is also phosphorylated in cells transformed by two mutants of RSV that induce spindle-shaped morphology (Rohrschneider and Rosok, 1983; Iwashita et ah, 1983). On the other hand, vinculin contains only a slightly elevated level of phosphotyrosine in the spindle-shaped cells transformed by PRCII virus (Sefton et al., 1981). We wished to know whether PRCIV virus or PRCIIp virus, both of which resemble PRCII virus in that they carry the fps gene but induce a polygonal rather than a spindle-shaped morphology in transformation, induced greater phosphorylation of vinculin on tyrosine. We have therefore determined the amount of phosphotyrosine in vinculin by labeling cells biosynthetically with ““Pi and isolating the protein by immunoprecipitation (Table 1). All src-containing viruses induced high levels of phosphorylation of vinculin on tyrosine, irrespective of the morphology of the transformed cells. Similarly, all of the fps-containing viruses induced only slightly increased phosphorylation of the protein on tyrosine, irrespective of the morphology of the transformed cells. Table 2 summarizes all transforming agents utilized and the parameters determined in the present study.

--.-.FIG. 3. Normal and transformed chick embryo fibroblasts (CEF) grown for 2 days to about 60% confluence. A given culture was doubly labeled by immunofluorescence for vinculin (panels A, C, E, G, I) and or-actinin (panels B, D, F, H, J, respectively). Pairs A, B: normal CEF; C, D: RSV-transformed CEF; E, F: Y73 virus-transformed CEF; G, H: PRC-IIp-transformed CEF; I, J: MC29-transformed CEF. Arrowheads in C and D indicate a condensed cluster of vinculin and cy-actinin patches (a “rosette”) while the arrows in C and D designate a region of spread-out patches. All panels, except for C and D, at same magnification; bars in C and I indicate 20 Fm.

60

NIGG

ET AL.

TABLE TYROSINE

Transforming

PHOSPHORYLATION

virus

OF VINCULIN

Oncogene

IN SEVERAL

TRANSFORMED

Phosphotyrosine“ (%I

CEF

Cell shape

15 14 17

Rounded Spindle-shaped Spindle-shaped

fPS

6

fPS fPS fps

7

4 5

Polygonal Polygonal Spindle-shaped Spindle-shaped

-

0.3

Flat

SR-RSV-A fFRsv F-l RSV

src src src

PRC-IIp PRC-IV PRC-II FSV None QPercentage

1

of total phosphoaminoacids

(phosphotyrosine,

DISCUSSION

Transformed fibroblastic cells exhibit quite different morphologies. Mouse fibroblasts transformed by benzpyrene and avian fibroblasts transformed by MC29 virus and methylcholanthrene are largely normal in morphology, cells transformed by MoSV, KiSV, and avian viruses carrying the fp.s gene have moderately altered morphologies, while cells transformed by RSV and Abelson virus are highly abnormal (Table 2). We have tried here to correlate changes in the number and distribution of adhesion plaques, in the organization of microfilament bundles, and in the extracellular deposition of fibronectin with these morphologies. Cell morphology is likely to be determined by multiple factors. The pronounced cell rounding induced by RSV is accompanied by reduced rates of synthesis of tropomyosin (Hendriks and Weintraub, 1981), vinculin (Sefton et al, 1981; Iwashita et al., 1983), procollagen (Levinson et al., 1975; Kamine and Rubin, 19’7’7),and fibronectin, by loss of fibronectin from the cell surface (Hynes, 1976; Olden and Yamada, 1977), by the phosphorylation of vinculin (Sefton et al, 1981), by the direct interaction of p60sK with adhesion plaques (Rohrschneider 1980; Nigg et ak, 1982) and cell to cell junctions (Shriver and Rohrschneider, 1981), and by a reduced number of adhesion plaques and microfilament stress

phosphoserine,

and phosphothreonine).

fibers (David-Pfeuty and Singer, 1980;Nigg et al, 1982). Finally, the interaction between microtubules and intermediate filaments also appears to be disrupted (Ball and Singer, 1981). All of these factors may contribute to the morphology of the transformed cell. In the transformed mouse cells examined here, we found a fairly good correlation between the extent of dissolution of intracellular adhesion plaques and microfilament bundles, the reduction of extracellular fibronectin, and the extent of cell rounding (Fig. 1). The cells possessing moderately altered morphologies had largely intact cytoskeletons and extracellular matrices. The highly altered RSVand Abelson virus-transformed cells eontained few adhesion plaques and stress fibers, and little fibronectin was apparent on their surfaces. Some of the transformed chick cells showed a similar correlation (Figs. 2 and 3). The polygonal cells produced by infection with PRCIV or PRCIIp virus contained a moderately reduced number of adhesion plaques and microfilament bundles and a moderately reduced amount of fibronection on their surfaces. In contrast, the highly rounded cells transformed by wild-type RSV contained very few adhesion plaques and microfilament bundles and little if any fibronection on their surfaces. The spindle-shaped cells produced by infection with fusiform variants of RSV and

polygonal

++ t-1 ++ t-1

+ +

src

abl

src

src

Mouse/SR RSV Mouse/AbMuLV

Chick/ffRSV

Chick/F1

RSV

’ FA, focal adhesions. b n.d., not determined. ’ Corroborated by surface iodination

Chick/PRC-II

experiments.

f Ps

+ (+)

+

src

RSV

Chick/SR

+

Spindle shaped

rounded

+ +

wc

Highly

+ -

(+) (-)

++ t-1

+ (+++)

+ (+++)

+++

+++

(-) (+)

fPS

+++ +++

Chick/MC29

-

+

Mouse/KiMSV

yes

(-)

(-)

(-)

Chick/Y73 Chick/PRC-Up/IV

+++

+++

++++

Presence of FA (rosettes)O

mos ras Polygonal/rounded

Slightly

Flat

Cell shape

Mouse/MoMSV

-

n.d. b -

-

Tyrosine-kinase activity

large T

gene

Mouse/SV40

?

?

QuaWmethylcholanthrene

-

Transforming

Mouse/benzpyrene

Mouse/-

Chick/-

Cell type and transforming agent

2

MOLECULARANDSTRUCTURALCORRELATIONSWITHCELLMORPHOLOGY

TABLE

+

+++

+++

+++

+

++++c

++++ ++=

+

+c +

+

++

++ ++++=

++c

++

++

+++

Abundance of fibronectin

++

++

++

++

+++

++++

Presence of stress fibers

ii 2

i

E q B

E

5 B

g

9

z

62

NIGG

ET AL.

terminant of cell morphology, it might be by PRCII virus-which carries a deleted form of the fps gene (Wong et al., 1982; expected to correlate with the extent of cell rounding. This is clearly not the case. Our Duesberg et al, 1983; Carlberg et ak, 1984)were unusual in that they contained abunobservations, and those of others (Iwashita dant cell surface fibronectin. In the case of et al., 1983; Rohrschneider and Rosok, 1983; PRCII virus-transformed cells, we found a Antler et al, 1985) suggest that the extent normal level of fibronectin. It may be that of tyrosine phosphorylation of vinculin is these cells are spindle-shaped, rather than primarily a property of the viral transpolygonal or round, because the synthesis forming gene and is not correlated with and accumulation of fibronectin is not re- transformed cell morphology. All cells duced to the extent it is in other transtransformed by viruses carrying the src which is highIy formed cells. A similar idea has been ad- gene contain vinculin vanced by Iwashita and colleagues to ex- phosphorylated on tyrosine, irrespective of plain the fusiform morphology of cells whether the cells are round or fusiform. Vinculin in all cells transformed by viruses infected by the d15 mutant of RSV (Iwashcarrying the fps gene contains only a low ita et al., 1983). In this regard, it is worth level of phosphotyrosine, irrespective of noting that Yamada et al. have shown that whether the cells are polygonal or spindlethe addition of large amounts of exogenous shaped. It should be noted that we have fibronectin to a variety of round transfound invariably that vinculin is phosformed cells causes them to assume a more elongated, fibroblastic morphology (Yaphorylated on tyrosine to a low but meamada et al., 1976). Our estimates of the surable extent in cells transformed by viamount of fibronectin on the surface of ruses carrying the fps gene (Table 1, and PRCII virus-transformed cells differ from Sefton et ah, 1981). In this respect our rethose of Guyden and Martin (Guyden and sults differ from those of Antler et al. (1985) Martin, 1982). We cannot explain this diswho detected no phosphotyrosine in vincrepancy. We found high levels of fibroculin in cells transformed by fps-containing nectin by both immunofluorescence and cell viruses. We cannot explain this discrepsurface iodination. ancy. While a reduction in vinculin-containing While the present results lend no support focal adhesions was generally found to for the view that vinculin phosphorylation correlate with the extent of cell rounding, is directly involved in the disruption of fothe reorganization of vinculin, actin and cal adhesions in cells transformed by tya-actinin into rosette-like structures was rosine kinase-encoding viruses, neither do only seen in cells transformed by RSV, they rule out this possibility. As discussed Abelson virus, PRCIIp, PRCIV, and Y73 previously (Sefton et aZ., 1981), only about virus, all of which encode transforming 1% of the vinculin molecules isolated from proteins with tyrosine protein kinase ac- RSV-transformed cells contain phosphotivity. These rosette-like structures are tyrosine. However, because phosphotyroprobably not merely focal adhesions that sine on vinculin may turn over rapidly, the have been relocated to a different part of steady-state level of this modification is not the cell surface. By interference reflection necessarily the quantity that is structurmicroscopy, they look more like close conally relevant. This might explain the lack tacts than focal adhesions and may provide of correlation of the steady-state level of considerably weaker anchorage than true vinculin phosphorylation with cell shape. focal adhesions (David-Pfeuty and Singer, Alternatively, it is entirely possible that 1980; Nigg et al., 1982). besides vinculin there may be other comVinculin is a substrate of a number of ponents associated with fibroblast focal viral transforming proteins which possess adhesion plaques (e.g., Maher and Singer, tyrosine protein kinase activity (Sefton et 1983; Burridge and Connell, 1983), or the ab, 1981). If the extent to which vinculin is cytoskeleton (Ball and Singer, 1981), that phosphorylated on tyrosine is a major de- can serve as additional substrates for ty-

FIBRONECTIN

IN TRANSFORMED

rosine kinases, and whose tyrosine phosphorylation may contribute to cytoskeletal disturbances and cellular shape changes. In summary, transformed fibroblasts exhibit a wide range of morphologies depending on the transforming agent. In general, the degree of change in morphology from that of the normal fibroblast correlates with changes in their intracellular filament networks, in their adhesions to the substratum, and in the structure of their extracellular matrices. In finer detail, however, it is likely that many factors, altered to different extents by different transforming agents, affect cell morphology. ACKNOWLEDGMENTS This work was supported by United States Public Health Service Research Grants CA 13213, CA 2977’7, CA 17289, and CA 22031 awarded by the National Cancer Institute. Erich Nigg acknowledges support from the Swiss National Science Foundation and from the Damon Runyon-Walter Winehell Cancer Fund. S. J. Singer is an American Cancer Society Research Professor. The authors thank Margie Adams and Cynthia Blais for excellent technical assistance and Glennis Harding for patient and expert help with the manuscript. We also thank Dr. C. D. Little, University of Virginia, for a kind gift of anti-flbronectin antibodies. REFERENCES AARONSON, S. A., and TODARO, G. J. (1968). Basis for the acquisition of malignant potential by mouse cells cultivated in vitro. Science 29,1024-1026. AARONSON, S. A., and WEAVER, C. A. (1971). Characterization of murine sarcoma virus (Kirsten) transformation of mouse and human cells. J. Gen ViroL 13,245-252. ABERCROMBIE, M., and DUNN, G. A. (1975). Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Exp. Cell Res. 92,57-62. ABERCROMBIE, M., HEAYSMAN, .I. E. M., and PEGRUM, S. M. (1971). The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Ezp. Cell Res. 67,359-367. ANDERSON, D. D., BECKMANN, R. P., HARMS, E. H., NAKAMURA, K., and WEBER, M. J. (1981). Biological properties of “partial” transformation mutants of Rous sarcoma virus and characterization of their pp60’* kinase. J. ViroL 37,445-458.

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