Proc. NatL Acad. Sci. USAVol. 78, No. 11, pp. 6986-6990, November 1981Cell Biology

Association of microtubules and intermediate filaments mi normalfibroblasts and its disruption upon transformation by atemperature-sensitive mutant of Rous sarcoma virus

(cytoskeleton/vimentin/tubulin/double immunofluorescence)

ERIC H. BALL* AND S. J. SINGERDepartment of Biology, University of California, San Diego, La Jolla, California 92093

Contributed by S . J. Singer, August 19, 1981

ABSTRACT By double indirect immunofluorescence, usingprimary rabbit antibodies to tubulin and guinea pig antibodies tovimentin, we have simultaneously labeled microtubules and in-termediate filaments in several types of cultured normal fibro-blasts. With well-spread interphase cells there was an extensivebut not complete correspondence of the labeling patterns for thetwo filamentous structures out to the cell periphery. This corre-spondence existed both at a gross level, where parallel but notcoincident arrays of thickly labeled strands of the two types of fil-aments were observed, and at a fine level, where thinly labeledstrands of the two were superimposed. The results suggest thatthere may be some type(s) of molecular linkages between micro-tubules and vimentin intermediate filaments that is under meta-bolic control. With NRK fibroblasts infected with a temperature-sensitive mutant (LA23) of Rous sarcoma virus, cells grown at thenonpermissive temperature (39C) showed the correspondence ofthe distributions of the microtubules and intermediate filamentscharacteristic of the normal phenotype but within 1 hr after a shiftto the permissive temperature (330C) there was an extensive re-traction of the intermediate filaments around the cell nucleuswhereas the microtubules remained dispersed into the cell pe-riphery. These results suggest that one ofthe functions carried outby p6Osrc, the protein kinase responsible for transformation byRous sarcoma virus, may be to modify the component(s) involvedin the putative linkages between microtubules and intermediatefilaments in the normal cells.

Three chemically distinct filamentous systems constitute thecytoskeleton of vertebrate cells: microfilaments, intermediatefilaments, and microtubules. The dynamics of these individualsystems and the interactions ofthese systems with one anotherprobably are of central importance to cellular functions such asintracellular transport, contractility, motility, and adhesion. Inthis paper, we are concerned with the possible interactions ofintermediate filaments with microtubules in. normal interphasefibroblasts and the effects of transformation by Rous sarcomavirus (RSV) on such interactions.

Although it appears that microfilaments structurally arelargely independent of the other two filamentours systems infibroblasts and other cultured cells (however, see ref. 1), thereis a growing body of evidence that at least certain types of in-termediate filaments may interact structurally with microtu-bules. Indirect evidence to-this effect is that several drugs thatspecifically cause the disruption of microtubules also induce amarked redistribution of desmin-type (2-4) and vimentin-type(5-7) intermediate filaments in muscle and fibroblast cells, re-spectively. Direct evidence for an interaction has been obtainedby electron microscopy in certain instances. In some electron

micrographs of cross sections of cell processes of cultured fi-broblasts (8), intermediate filaments are arranged in a hexagonalarray around individual microtubules. In other micrographs oflongitudinal thin sections of fibroblasts and epithelioid cells(9-11), occasionally an individual intermediate filament is ob-served aligned in close proximity and parallel to a microtubuleover a considerable distance, and even linkage elements appearbetween them. On the other hand, such observations are cer-tainly not the rule; in most cases, apparently unassociated in-termediate filaments and microtubules are observed. The na-ture and significance of intermediate filament-microtubuleinteractions has therefore not been clear.

Double immunofluorescence experiments, despite theirlower resolution compared to electron microscopy, can be use-ful in providing an overview of the relative dispositions of twocytoskeletal systems in well-spread cells. An important aspectof the technique is that, in appropriate circumstances, evensingle filamentous structures such as individual cytoplasmicmicrotubules (12) can be visualized. This is because what isvisualized is a divergent fluorescent beam emanating from theimmunolabeled filament. In an earlier report from this labo-ratory (4), it was demonstrated by double indirect immunoflu-orescent analysis in chicken gizzard smooth muscle cells thatthe distributions of desmin-type intermediate filaments andmicrotubules showed a significant degree of correlation ex-tending out to the cell periphery.

In this paper, we report similar experiments in cultured fi-broblasts to study the relative distributions of vimentin-typeintermediate filaments and microtubules in the same cells andto study the effects of transformation by RSV on these relativedistributions. The use of cells infected with a temperature-sen-sitive RSV mutant has revealed a transformation-correlatedchange in the intermediate filament/microtubule distributions.

MATERIALS AND METHODSAntigens and Antibody Preparations. Vimentin was purified

from BHK cells as reported (13). An intense band at 58,000daltons was observed on Coomassie-stained NaDodSO4/poly-acrylamide gels, and a faint band was observed at about 300,000daltons (Fig. 1, lane b). Guinea pig antisera were raised to vi-mentin by primary intradermal injections of20 ,g in completeFreund's adjuvant, followed 2 weeks later with a booster injec-tion of 2 ug in incomplete Freund's adjuvant (preimmune seradid not show any filamentous staining). The antivimentin an-tibodies were affinity purified on an immunoadsorbent pre-pared by dialyzing purified vimentin into 0.1 M potassium

Abbreviation: RSV, Rous sarcoma virus.*Present address: Department of Biochemistry, University ofWesternOntario, London, Ontario, Canada N6A SC1.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Proc. NatL Acad. Sci. USA 78 (1981) 6987

ab c dFIG. 1. Electropherograms of NaDodSO4/8% polyacrylamide gel

electrophoresis. Lanes: a, molecular weight standards (from top to bot-tom)-filamin (240,000), 0-galactosidase (130,000), phosphorylase A(95,000), bovine serum albumin (68,000), catalase (60,000), IgG heavychain (50,000), and actin (43,000); b, purified preparation of vimentinfrom BHK cells, showing a major Coomassie-stained- band at 58,000daltons and a minor band at about 300,000; c, autoradiograph of an

immunostained nitrocellulose paper (transfer was effectedfrom the gelof an electrophoretic separation of a whole cell extract of NRK cellsto the nitrocellulose paper and the paper was then treated with guineapig antivimentin antibodies and 1"I-labeled protein A; only a singleband with the mobility of vimentin was labeled); d, a control for theexperiment in lane c, which was carried out identically except thatnormal guinea pig IgG was substituted for antivimentin antibodies.These results demonstrate the monospecificity of the antivimentinantibodies.

phosphate buffer (pH 9.0) and then mixing it with glutaralde-hyde-activated Ultrogel AcA22 (14) for 18 hr at room temper-ature. To test for the specificity of these antibodies, whole ex-

tracts of NRK cells were subjected to NaDodSO4/polyacrylam-ide gel electrophoresis, and transfers were then made fromthese gels to nitrocellulose paper (15). The paper was thentreated with the affinity-purified antibodies to vimentin fol-lowed by "2I-protein A. The results (Fig. 1, lanes c and d)showed that only the vimentin band was immunolabeled.

Affinity-purified rabbit antibodies to chicken brain tubulin(16), affinity-purified and cross-adsorbed goat antibodies to rab-bit and guinea pig IgG, and the preparation of fluorescein andrhodamine conjugates of the goat antibodies (4) were as

described.LA23-NRK cells and uninfected NRK cells were the gifts of

Peter K. Vogt, and the BALB 3T3 cells were provided by TonyHunter. Cells were cultured for a minimum of2 days, fixed with3% formaldehyde, made permeable with Triton X-100, im-munolabeled, and examined in a Zeiss Photoscope III instru-ment as described (4, 16).

RESULTS

LA23-NRK Cells at 390C (Normal Phenotype). NRK cellsinfected with LA23, a temperature-sensitive mutant of RSV,and grown in monolayer culture at 39°C exhibit a well-spreadappearance characteristic ofthe normal phenotype. Double im-munolabeling of such cells for tubulin and vimentin (Fig. 2 Aand B and Fig. 3 A and B) showed a strong correspondence of

the distributions of the microtubules and intermediate fila-ments out to near the cell periphery in these interphase cells.This correspondence was of two kinds, gross and fine. At thegross level, the arrays of microtubules and intermediate fila-ments, although not precisely coincident, were remarkably par-allel and where gaps existed in the distribution ofthe one, theywere likewise present in the other. From the Nomarski imageof these cells (Fig. 2C) there was no indication of any surfaceinhomogeneities that would correspond to the gaps in the fil-ament distributions. In addition, at the fine level, there werelightly labeled strands of microtubules that were coincidentwith lightly labeled strands ofintermediate filaments (e.g., cor-responding arrowheads in Figs. 2 A and B and 3 A and B).

Other Normal Fibroblasts. In order to explore the possiblegenerality of these findings, we examined other normal fibro-blasts by the same double immunofluorescent labeling exper-iments. With human WI-38 cells, there was also a striking de-gree ofcorrespondence (Fig. 4A and B) ofthe microtubules andintermediate filaments out to the cell periphery.On the other hand, the 3T3 cells exhibited a distribution of

intermediate filaments that was largely perinuclear (Fig. 4D)with only partial extension into the peripheral areas of the cellwhere microtubules were present (Fig. 4C). Uninfected NRKcells showed a degree of correlation of microtubule and inter-mediate filament distributions (Fig. 4 E and F) that was in be-tween that of the WI-38 and the 3T3 cells. The intermediatefilaments extended further into the cell periphery than in the3T3 cells but not as far as in the WI-38 cells or in the LA23-NRKcells grown at 390C.LA23-NRK Cells at 33TC (Transformed Phenotype). When

cultured at 330C LA23-NRK cells exhibit the transformed phe-notype. Morphologically, they are generally more rounded thanthe same cells grown at 390C. In order to compare cells of dif-ferent phenotypes, examples were chosen from cells grown at330C and 390C in which the degree of cell spreading and ofmicrotubule extension were similar (Fig. 3). Another exampleof an LA23-NRK cell cultured at 39°C is shown in Fig. 3A andB. At 24 hr after a shift of these cells from 390C to 330C, theintermediate filaments (Fig. 3F) were largely retracted aroundthe nucleus relative to the microtubules (Fig. 3E). Most im-portant (see Discussion), this relative retraction of the inter-mediate filaments from the microtubules was already fully ap-parent only 60 min after the shift from 390C to 330C (Fig. 3 Cand D).

DISCUSSIONIn the peripheral regions ofseveral types ofwell-spread normalfibroblasts in interphase, we have observed by double indirectimmunofluorescence experiments an extensive correspon-dence of the distributions of microtubules and vimentin-typeintermediate filaments similar to the correspondence previ-ously observed (4) for microtubules and desmin-type interme-diate filaments in chicken gizzard smooth muscle cells. Controlexperiments (not shown) ofthe same type as used in the earlierstudy (figure 2 of ref. 4) demonstrated that this correspondencecould not be due to optical or immunolabeling artifacts. Thecorrespondence was greater when the fibroblasts were morespread, as with LA23-NRK cells grown at 39°C and WI-38 cellscompared to the uninfected NRK and BALB 3T3 cells.One aspect of this correspondence is an extensive codistri-

bution, but not coincidence, of strongly labeled strands of mi-crotubules and of intermediate filaments. For several reasons,we believe these codistributions to be significant and specificand not due, for example, to a copacking of these filaments byexclusion from adjacent portions ofthe cell. First, the Nomarskiimages ofthese cells do not show surface undulations that would

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6988 Cell Biology: Ball and Singer

FIG. 2. Double indirect immunofluorescent labeling of an LA23-NRK cell, grown at 39TC (normal phenotype), for tubulin (A) and vimentin(B) in the same focal plane. For tubulin labeling, treatment with affinity-purified rabbit antibodies to chicken brain tubulin was followed by flu-orescein-conjugated affinity-purified goat antibodies to rabbit IgG; for simultaneous vimentin labeling, affinity-purified guinea pig antibodies toBHK vimentin was used, followed by rhodamine-conjugated affinity-purified goat antibodies to guinea pig IgG. Arrowheads in A and B point toa superimposed lightly labeled microtubule strand and vimentin intermediate filament strand. (C) The same cell in Nomarski optics is shown. (Allfields at same magnification; bar in C represents 20 pum.)

correspond to the simultaneous gaps in the distributions of thetwo filament systems. Second, although not shown here, thedistributions ofactin-containing microfilaments do not correlatewith those of microtubules (17, 18) or intermediate filamentsin similar cells. Third, in other types of well-spread culturedcells containing pre-keratin-type intermediate filaments, dou-ble immunofluorescent labeling for these filaments and formicrotubules (1) revealed both to be spread throughout the cellperiphery but did not exhibit any correspondences betweenthem at either the gross or fine levels such as are shown in Figs.2 and 3 A and B (see also ref. 19).

In addition to the gross correspondence of thicker strands ofthe two filament types, there were significant numbers of in-stances (examples are designated by arrowheads in Figs. 2A andB and 3 A and B) in which lightly labeled microtubule and vi-mentin intermediate filament strands were coincident over aconsiderable distance. In the case of tubulin immunolabeling,it has been shown directly that the most lightly labeled strandscorrespond to individual microtubules (12). In these instances,therefore, the suggestion is that single microtubules and single(or a few) intermediate filaments may be codistributed. Suchcoincident distributions at the low resolution of the light mi-croscope may correspond to the close lateral appositions be-tween single microtubules and one or a few intermediate fila-ments occasionally seen in electron micrographs of culturedcells (8-11).A reasonable explanation of the correspondence of the two

filament distributions is that there exist one or more types ofmolecular linkages between them. If such linkages occurred ataperiodic intervals along the lengths of the two filaments, thesizes of the intervals could vary widely. If the intervals wereshort, the two filaments would be individually apposed, thusaccounting for their occasional coincidence over considerabledistances. Ifthe intervals were long, however, the two filamentswould be only sporadically linked, and bundles of one filamenttype might be aligned loosely parallel with bundles ofthe other.This could account for the gross correspondence of the two dis-tributions. The proposition that microtubules and intermediatefilaments may be linked together has already been made and

given credence as a result of electron micrographs which showoccasional bridges between the two inside cells (cf. figure 3Ain ref. 11). The nature of the components forming these mor-phological bridges is not known. One or more proteins that areassociated with intermediate filaments or microtubules or both(refs. 20-22; see also ref. 23) conceivably might perform sucha function.Any such linkages between microtubules and intermediate

filaments would presumably be subject to the metabolic controlofthe cell. During mitosis ofnormal fibroblasts, the distributionof vimentin labeling extends throughout the rounded cell,whereas that of tubulin is confined to the spindle (not shown;see ref. 7), suggesting dissociation of the two structures. Theformation of such linkages may also depend on the extent ofcellspreading in normal interphase cells. This would account for thevariation in.the extent of the correspondence for different cellsshown in Figs. 2 and 4. Furthermore, the linkages are likely tobe disrupted when specific microtubule-disaggregating drugssuch as Colcemid or vinblastine are added to cells, thus ex-plaining the observations (not shown: see refs. 7 and 19) that,in addition to the microtubules being disorganized, the vimen-tin intermediate filaments are significantly redistributed aftersuch treatments.

Transformation of fibroblasts by RSV also appears to disso-ciate the microtubules and intermediate filaments in these cells.This transformation is due to the action of the protein kinasep6O'rc which is coded for by a single gene of the virus. This ki-nase causes the phosphorylation of tyrosine residues of a smallnumber ofas yet largely undefined protein substrates inside thetransformed cell (24). The rounding of the RSV-transformedcells has led to the suggestion that elements ofthe cytoskeletonmay be substrates of p6Y'r. One such substrate appears to bevinculin (24), and phosphorylation of its tyrosine has been im-plicated in the disorganization of the microfilament bundles in-side the normal cells (25). In this article, we have demonstratedthat another consequence of RSV transformation is the loss ofthe correspondence of the distributions of microtubules andvimentin intermediate filaments characteristic of the normalcell. Whereas the microtubules remain extended to the cell

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'-

FIG. 3. Double indirect immunofluorescent labeling of LA23-NRK cells grown at 39TC (A and B), and at 1 hr (C and D) and 24 hr (E and F)after a shift from 390 to 3300. (A, C, and E) Immunolabeled for tubulin; (B, D, and F) Immunolabeled for vimentin (see Fig. 2 for procedures).Arrowhead in A and B points to a superimposed lightly labeled microtubule strand and intermediate filament strand. (All fields at same magni-fication; bar in A represents 20 gm.)

periphery of the generally rounded transformed cell (Fig. 3 Cand E), the intermediate filaments are retracted around thenucleus (Fig. 3 D and F). That this change is a direct result oftransformation is indicated by the fact that it is fully expressedwithin 1 hr after a shift of the LA23-NRK cells from nonper-missive to permissive temperature, and is already seen in a sub-stantial fraction of the cells within 30 min (not shown). It istherefore among the most rapid phenotypic changes observedin such temperature-sensitive virus-infected cells.

In addition to these experiments with the LA23-NRK cells,we have also carried out similar studies comparing uninfectedNRK cells and ones infected and transformed with a wild-typestrain of RSV, B77. Although the comparison is made moredifficult by the extensive rounding of the B77-NRK cells, itcould be shown that in the transformed cells the vimentin in-termediate filaments were retracted into a perinuclear distri-bution relative to the microtubules, compared with the distri-butions in NRK cells themselves (1).

Experiments related to those presented in this article havebeen carried out (7) with separate lines of a NIL8 hamster celland a derivative infected with a hamster sarcoma virus. In theseinfected cells, the intermediate filaments were observed to be

in a perinuclear distribution, compared to a more extendeddistribution in the uninfected cells. These experiments, how-ever, differed in two significant respects from ours. First, onlysingle immunofluorescence labeling for either tubulin or vi-mentin was carried out in a given cell population, and thereforeno significant assessment of the correspondence of the distri-butions ofmicrotubules and intermediate filaments in the samenormal cell could be made. Second, a temperature-sensitivevirus was not used. In view of the fact that agents other than,and unrelated to, transforming viruses also induce a change toa perinuclear distribution of intermediate filaments in fibro-blasts (26), it is not clear from these hamster cell experimentswhether the effects observed were due to transformation perse or to some secondary metabolic differences between the twocell lines used. The observations made with the hamster celllines, however, are clearly consistent with ours.The results reported in this paper for well-spread normal fi-

broblasts in interphase have led to the suggestion that one ormore types of metabolically controlled linkages exist betweenmicrotubules and vimentin intermediate filaments. It followsthat our results with the cells infected and transformed by thetemperature-sensitive mutant of RSV lead to the further sug-

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P.I. I

FIG. 4. Double indirect immunofluorescent labeling of WI-38 cells (A and B), BALB 3T3 cells (C and D), and uninfected NRK cells (E and F).(A, C, and E) Immunolabeled for tubulin; (B, D, and F) same cells, respectively, immunolabeled for vimentin with the same reagents as used forFig. 2. (All fields at same magnification: bar in A represents 20 gm.)

gestion that one or more of these linkage components could bein vivo substrates for the p6Owc kinase activity.

We gratefully acknowledge the excellent technical assistance of Mrs.Margie Adams and Yun-Yun Chen. These studies were supported byU.S. Public Health Service Grants GM-15971 and CA-22031 to S.J.S;E. H. B. was a Postdoctoral Fellow of the Medical Research Council ofCanada, 1979-1981. S.J.S. is an American Cancer Society ResearchProfessor.

1. Singer, S. J., Ball, E. H., Geiger, B. & Chen, W.-T. (1981) ColdSpring Harbor Symp. Quant. Biol 46, in press.

2. Ishikawa, H., Bischoff, R. & Holtzer, H. (1968)J. Cell Biol 38,538-555.

3. Lazarides, E. (1978) Exp. Cell Res. 112, 265-273.4. Geiger, B. & Singer, S. J. (1980) Proc. Natl Acad. Sci. USA 77,

4769-4773.5. Goldman, R. D. (1971) J. Cell Biol 51, 752-762.6. Goldman, R. D. & Knipe, D. M. (1972) Cold Spring Harbor

Symp. Quant. Biol 37, 523-534.7. Hynes, R. 0. & Destree, A. T. (1978) Cell 13, 151-163.8. Goldman, R. D. & Follett, E. A. C. (1969) Exp. Cell Res. 57,

263-276.9. Wang, E. & Goldman, R. D. (1978) J. Cell Biol 79, 708-726.

10. Franke, W. W., Grund, C., Osborn, M. & Weber, K. (1978) Cy-tobiologie 17, 365-391.

11. Borenfreund, E., Schmid, E., Bendich, A. & Franke, W. W.(1980) Exp. Cell Res. 127, 215-235.

12. Osborn, M., Webster, R. E. & Weber, K. (1978)J. Cell Biol 77,R27-R34.

13. Starger, J. M., Brown, W. E., Goldman, A. E. & Goldman, R.D. (1978) J. Cell Biol 78, 93-109.

14. Ternynck, T. & Avrameus, S. (1976) ScandJ. Immunol Suppl 3,29-35.

15. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl Acad.Sci. USA 76, 4350-4354.

16. Heggeness, M. H., Simon, M. & Singer, S. J. (1978) Proc. NatlAcad. Sci USA 75, 3863-3866.

17. Heggeness, M. H., Wang, K. & Singer, S. J. (1977) Proc. NatlAcad. Sci. USA 74, 3883-3887.

18. Gottlieb, A. I., Heggeness, M. H., Ash, J. F. & Singer, S. J.(1979) J. Cel. Physiot 100, 563-578.

19. Osborn, M., Franke, W. W. & Weber, K. (1980) Exp. Cell Res.125, 37-46.

20. Pytela, R. & Wiche, G. (1980) Proc. Natl Acad. Sci. USA 77,4808-4812.

21. Lin, J. J.-C. & Feramisco, J. R. (1981) Cell 24, 185-193.22. Granger, B. L. & Lazarides, E. (1980) Cell 22, 727-738.23. Runge, M. J., Laue, T. M., Yphantis, D. A., Lifsics, M. R.,

Saito, A., Altin, M., Reinke, K. & Williams, R. C., Jr. (1981)Proc. Natl Acad. Sci. USA 78, 1431-1435.

24. Sefton, B. M., Hunter, T., Ball, E. H. & Singer, S. J. (1981) Cell24, 165-174.

25. David-Pfeuty, T. & Singer, S. J. (1980) Proc. Nati Acad. Sci. USA77, 6687-6691.

26. Sharpe, A. H., Chen, L. B., Murphy, J. R. & Fields, B. N. (1980)Proc Natl Acad. Sci. USA 77, 7267-7271.

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