Experimental Cell Research 118 (1979) 95-109

HELA CELLS CONTAIN INTERMEDIATE-SIZED FILAMENTS

OF THE PREKERATIN TYPE

WERNER W. FRANKE,’ ERIKA SCHMID,’ KLAUS WEBER* and MARY OSBORN*

IDivision of Membrane Biology and Biochemistry, Institute of Experimental Pathology, German Cancer Research Center, D-6900 Heidelberg, and 2Departmenf of

Biochemistry, Max Planck Znstitute for Biophysical Chemistry, D-3400 GBttingen, Germany

SUMMARY

Immunofluorescence microscopy using antibodies raised against protein constituents of the differ- ent tvoes of intermediate-sized filaments has shown that in HeLa cells filaments containing a prekeratin-like protein (cytokeratin) predominate. The wavy filament bundles decorated by antibodies against prekeratin are similar to those described in other cells of epithelial origin. These bundles of intermediate-sized (6-l 1 nm) filaments are also described by electron microscopy in intact cells and in cytoskeletal preparations obtained by cell lysis and extraction with low and high salt buffers and Triton X-100. The occasional occurrence of desmosome-attached tonotibrillar bundles of intermediate-sized tilaments is also shown. When HeLa cells are treated for long times with colcemid to induce perinuclear whorls of intermediate-sized filaments, these aggregates of filaments are strongly stained by antibody to vimentin, the major polypeptide of the intermediate- sized filaments of murine 3T3 cells. However, they are not stained by antibody to prekeratin, and the display of the prekeratin-containing tonotilament-like structures is similar to that seen in untreated cells. SDS-polyacrylamide gel electrophoresis of cytoskeletons prepared under condi- tions in which intermediate-sized filaments are retained show the presence of a polypeptide which co-migrates with one component of bovine prekeratin, and a-second polypeptibe- which co- migrates with vimentin nurified from mouse 3T3 cells. The data show (i) that two different types of in&mediate-sized filaments can be present in the same cell and can be distinguished immunologi- cally; and (ii) that the expression of a prominent epithelial structural marker, i.e. prekeratin- containing filaments, can be maintained in malignancy and continuing proliferation in vitro.

Intermediate-sized filaments have been as- sumed to represent a single class of cyto- skeletal fibers (for references on similarities see, e.g. [l-15]). Recently, based mainly on immunological criteria, it has become clear that in contrast to microfilaments and microtubules the intermediate-sized lila- ments can be divided into several subgroups (e.g. [16-18, 193). In the course of con- tinuing studies on the nature and immuno- logical cross reactivity of the intermediate- sized filaments present in various cultured cells antisera have been developed which

7-781X02

when used in immunofluorescence micro- scopy discriminate at least three of the sub- groups of intermediate-sized filaments. (a) The tonolilament-like intermediate lila- ments characteristic of epithelial cells, in- cluding epidermal cells, rat kangaroo PtKl and PtK2 cells, human kidney epithelial cells, and mammary gland epithelial cells, are identified either by rabbit auto-immune sera [lo] or by guinea pig antisera to pre- keratin [ 171. (b) The intermediate-sized filaments characteristic of muscle tissues, including heart muscle cells in culture, are

96 Franke et al.

Fig. 1. Arrays of bundles of intermediate-sized fila- meshwork architecture of these fibril arrays (e.g. a, c), ments present in relatively well spread cells of a HeLa their exclusion, in these cells, from the flattened cell S3-derived clone growing in monolayer culture, re- periphery (denoted by arrowheads in (I, b, d), their vealed by indirect immunofluorescence microscopy frequent correspondence in adjacent cells (b), and using guinea pig antibodies against purified bovine their tendency to exhibit radial orientations (d). Bars hoof prekeratin (cf [17]). Note the three-dimensional in these and all subsequent light micrographs, 20 pm.

identified with sera against desmin [16]. group has been identified using antibodies (c) The intermediate-sized filaments char- prepared against polypeptides of molecular acteristic of cells of mesenchymal origin, in- weight approx. 57000 isolated either from eluding mouse 3T3 cells, rat and hamster cytoskeletons of mesenchymal-derived fibroblasts, which are also found as perinu- cells in culture [14, 181 as well as using a clear whorls induced by treatment with col- particular rabbit serum [13]. We have pro- cemid, represent another class. This sub- posed elsewhere the name “vimentin” for

Keratin in 100 A-filaments of HeLa cells 97

Fig. 2. Arrays of tibrils decorated by antibodies against prekeratin in a monolayer culture of HeLa S3 cells dif- ferent from those shown in fig. 1 (less flattened cell type). (a) Survey of a dense culture; (b) corre-

the polypeptide subunit of this latter class of intermediate-sized filaments [ 181.

The purpose of this communication is to characterize the classes of intermediate- sized filaments present in HeLa cells. Im- munofluorescence microscopy with anti- body against prekeratin reveals elaborate arrays of tonofilament-like filaments which can also be documented by electron micro- scopy. In addition, the perinuclear whorls induced by colcemid treatment of HeLa cells can be decorated by antibody to vi- mentin, but not by antibody to prekeratin. These findings seem of importance, not only because HeLa is the classic prototype of a cell which is highly malignant and capable of unlimited growth in culture, but also because it shows that tumour cells ori- ginally derived from epithelial tissue [35] still retain the specialized prekeratin type of intermediate-sized filaments, typical of this tissue type. Thus the possibility arises that identification of tumour cells of un- known origin may in some instances be fa- cilitated by the use of specific antibodies against the different subgroups of inter- mediate-sized filaments.

spondence of arrays of prekeratin-containing fibrils in adjacent cells, suggestive of tonotibril-like intercel- lular associations at desmosome-equivalent struc- tures.

MATERIALS AND METHODS

Cells HeLa cells were obtained from the American type cul- ture collection (CCL 2) via Flow Laboratories (Bonn). Both the original culture and various subclones were tested. HeLa S3 (CCL 2.2) was obtained from Cold Spring Harbor Laboratory (courtesy of Dr T. Grod- zicker) and from the Salk Institute (courtesy of Dr W. Deppert) as well as from Flow Laboratories. In ad- dition, several of the HeLa cell clones growing in monolayer culture previously described [22] were ex- amined. Some of the HeLa S3 cultures were also ex- amined after growth in suspension. HeLa cells from all sources behaved equivalently in the tests described here. The cells were grown in standard cell culture medium supplemented with 10% calf serum. For the induction of perinuclear aggregates of intermediate filaments cell cultures were treated with 10m6 M col- cemid for the times indicated.

Antibodies and indirect immuno- fluorescence microscopy The following antibodies were available. (I) Antisera raised in guinea pigs against purified bovine hoof pre- keratin polypeptides were used either as the IgG frac- tion, or were used after being made monospecific by passage over prekeratin covalently coupled to Sepharose 4B (see [17, 181). (2) Antisera raised in guinea pigs against vimentin, purified from high salt- extracted cytoskeletons of mouse 3T3 cells (for fur- ther details see [18]). Controls with the preimmune sera were neeative. Antisera were used after 15-fold dilution into phosphate-buffered saline (PBS). Mono- specific IgGs were used at 0.05 mg/ml. The second antibody, fluorescein-labeled goat anti-guinea pig IgGs, was from Miles-Yeda (Israel) and was used at a 1 : 15 dilution. (3) Parallel experiments involved guinea

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pig non-immune sera and pre-immune sera as controls. In addition. rabbit IgGs against actin TIO. 23. 241,

I I

against tubulin [ 10, 25, 261, and against rat liver micro- somal cytochrome b, [27] all of which have been docu- mented elsewhere. The latter antibody served as a membrane marker [27].

For indirect immunofluorescence microscopy cells were grown either on glass coverslips, or were grown in suspension and allowed to attach for very short time intervals to a glass coverslip. They were then fixed using organic solvents only [lo] and processed for im- munofluorescence microscopy as described elsewhere [lo, 17, 23-261.

Electron microscopy Cells grown on coverslips were fixed and processed for examination of ultrathin sections [12,28]. Pellets of ex- tracted cell preparations enriched in intermediate-sized filaments (see below) were prepared for ultrathin sec- tioning in a similar manner or were used directly for negative staining with either phosphotungstic acid or uranyl acetate as described [29].

Preparations enriched in intermediate- sized filaments The preparations of fractions from PtK2 cells en- riched in cvtoskeletal material bv combined extrac- tions in low-and high salt buffers and Triton X-100 has been described 117, 291. For HeLa cell lavers which adhere less tightiy to the dish the procedure was modi- fied as follows [ 181.

(i) Monolayers were rinsed twice in TNM-buffer (10 mM Tris-HCI, 140 mM NaCI, 5 mM MgCl,, pH 7.6). The monolayer cells were then incubated briefly (2-l min) in TN-buffer (10 mM Tris-HCI, 140 mM NaCl, pH 7.6) containing I % Triton X-100 at room temperature. Material remaining attached to the sub- stratum consisted mainly of nuclei and cytoskeletal elements (cf [15, 29-311). The cell residues were then incubated for 30 min at room temperature in TN-buffer containing 0.5 % Triton X-100 and 1.5 M KC1 (pH 7.6). The dispersed cell material was collected by 20 min centrifugation at 3 500 g. The pellet obtained consisted of residual nuclear structures and intermediate-sized filaments but also contained variable amounts of some actin-containing tibrillar material. This pellet material was washed twice by brief dispersion in IO mM Tris- HCI (pH 7.6) followed by centrifugation.

(II) In some experiments, this material was further incubated for 15-25 min in 10 mM Tris-HCI buffer (pH 7.4) containing 0.2 mM MgCI, and 100 wg/m] pancreatic deoxyribonuclease I (2839 U/mg, from Worthington Biochem. Corp., Freehold, N.J.) at room temperature in order to digest the DNA present in these cytoskeleton-enriched preparations. For ex- amination of the proteins by SDS-polyacrylamide gel electrophoresis precipitates obtained with 5% cold tri- chloroacetic acid were washed with acetone and taken up in sample buffer (see below; cf [17]).

(iii) Altemativelv, DNA was removed from the residual cytoskeletal material by the following pro- cedure. The cell material treated with TN-buffer con-

taining I c/c Triton X-100 (see above) was harvested and pelleted by centrifugation for IO min at 3 500 g. The pelleted material was then treated with DNAse- containing buffer as described above. and the dieestion was stopped by addition of 100 vol of TN-buff& con- taining I.5 M KCI and 0.5% Triton X-100. After in- cubation for 30 min the pellets obtained were washed with IO mM Tris-HCI buffer (pH 7.4).

{iv) Cell material enriched ‘in cytoskeletal elements was prepared as described under (ii), and high salt buf- fer (TN buffer containing I.5 M KCI and 0.5% Triton X-100) was added directly to the DNAse digest solu- tion. After incubation for another 20 min the residual material was pelleted by ultracentrifugation as de- scribed above.

Gel electrophoresis Procedures used for separation of polypeptides by gel electrophoresis in sodium dodecylsulfate (SDS)-con- taining polyacrylamide gels were as described [ 171.

RESULTS

Immunojluorescence microscopy of arrays of intermediate-sized filaments

Use of antibody against prekeratin reveals a system of wavy fibrous structures ex- tending throughout the cytoplasm (figs 1 and 2). In some HeLa cell cultures the more flattened portions of the cell periphery do not show these fibers (fig. la, b, d), in contrast to less spread HeLa cells in which the prekeratin-type fibrils extend into the cell periphery (fig. 2). The fibers run over and around the nucleus and in many cells appear to be more numerous in the peri- nuclear cytoplasm (e.g., fig. 1 c, d). Nearly radial arrangements of such fibrils are sometimes found, and when they occur they

Fig. 3. Reaction of HeLa cells grown in monolayer (same culture as presented in fig. 1) with guinea pig antibodies against murine vimentin, the major poly- peptide of the intermediate-sized filaments of mouse 3T3 cells (cf [18]), seen by indirect immunofluores- cence microscopy. Note only weak and indistinct fluorescence in normally growing cells (u), in contrast to the strong decoration of the perinuclear whorls of intermediate-sized filaments induced by prolonged treatment (24 h) with colcemid (b). The spotty fhrores- cence over nuclei sometimes seen in such experiments (e.g. 3~) is not specific and is not always observed.

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100 Franke et al.

are often limited to one side of the cell (fig. lb, d). These tibril arrays are clearly dif- ferent from those of actin-containing micro- filament bundles and of microtubules (for these patterns in HeLa cells see [32]; for ar- rangements of these structures in other cells see, for example [lo, 23-261). Adjacent cells sometimes show indications of mirror image symmetry of fibril arrangement (e.g., fig. lb). Fiber arrays suggestive of tonofibril- like correspondence are also seen (fig. 26, see also fig. 4a and below). These fibrous arrays are rearranged during mitosis, giving rise to a “basket” surrounding the mitotic spindle as described already for PtK2 cells [lo, 12, 171. This basket-like arrangement is also characteristic of HeLa cells grown in suspension. Decoration of HeLa cells with antibodies against vimentin, the structural protein of intermediate-sized filaments characteristic of mesenchymal and various other non-epithelial cells [18] shows only very faint background staining (fig. 3 a).

When HeLa cells treated for long time with colcemid are examined the charac- teristic drug-induced, petinuclear whorls of intermediate-sized filaments are decorated specifically by antibodies against vimentin (fig. 3 b) and are easily visualized in im- munofluorescence microscopy (fig. 3 b). When these colcemid-treated cells are ex- amined with antibodies to prekeratin, the perinuclear whorls seem not to be stained. Instead these cells still show a typical ex- tended display of fibers (fig. 4a-d; cf [lo]). In most cells the prekeratin-containing fibers now appear to border on the cell periphery (fig. 4b-d). A direct comparison of the fiber patterns of drug-treated cells seen after decoration either with antibodies to prekeratin (fig. 4a-d) or with antibodies to vimentin (fig. 4e) clearly illustrates the difference in arrangement of these two systems of intermediate-sized filaments.

Electron microscopy Intermediate-sized filaments of 6-11 nm have been described in intact HeLa cells by one of us [2] and in cytoskeletal prepara- tions of HeLa cells by Lenk et al. [33]. Simi- lar filaments can be seen in electron micro- graphs of other authors although they have not been specifically identified as inter- mediate-sized filaments (e.g. [34]; cf [35]). In the context of this study, we have re- examined the display and the ultrastructure of the intermediate-sized filaments present in HeLa cells grown in monolayer culture. In ultrathin sections through surface- attached, non-treated HeLa cells typical wavy arrays of intermediate-sized filaments are recognized (fig. 5a). In many cells the frequency of such filaments appears greatly enhanced in the immediate vicinity of the nuclear envelope (cf [2]). Intermediate- sized filaments can occur either as single filaments or, more characteristically, as ag- gregated filament bundles (fig. 5a) remi- niscent of the bundles which we have de- scribed in other epithelia-derived cells in culture, including PtK2 cells (e.g., [lo, 12, 171). The diameters and the frequency of such bundle aggregates of intermediate-

Fig. 4. HeLa cells grown in monolayer culture (same type of culture as shown in fig. 1) and treated with col- cemid show relatively extended meshwork arrays of bundles of intermediate-sized filaments of the pre- keratin type as revealed by immunofluorescence microscopy using antibodies against prekeratin (a-d). In such somewhat retracted, rounded off cells these fibril bundles now border on the cell periphery (a-d), and the perinuclear accumulation of such fibrils seems to be stronger in many cells (6, c). Pronounced radial orientations of frbrils of the prekeratin-like type are especially frequent in a subpopulation of cells that are unusually flat and occupy large areas (4. Oc- casionally, desmosome-tonofibril-like cell-to-cell cor- respondences can still be recognized (e.g., the cells in- dicated by the arrow in the insert in a). (e) For direct comparison, the decoration, in the same cells (same culture dish, different coverslips), with antibodies to murine vimentin which is exclusively found in the mas- sive petinuclear whorl-like aggregates of intermediate- sized filaments.

Keratin in 100 .d-filaments of HeLa cells 101

102 Franke et ul.

sized filaments are somewhat variable from cell to cell, perhaps reflecting differences in the cell cycle and cell density. Very oc- casionally, an association of such bundles of intermediate-sized filaments with desmo- some-like structures has been observed (fig. 4b). This finding is a further strong indica- tion for the tonolilament-like character of these filaments.

When HeLa cells are treated for long times with colchicine or colcemid, the dis- play of the tonofdament-like bundles does not change significantly (fig. 4c), in agree- ment with the results seen in immuno- fluorescence microscopy (cf fig. 4a-d). However, under such conditions extensive whorl-like aggregates of relatively loosely packed intermediate-sized filaments are formed in the perinuclear cytoplasm (fig. 6a-c; see also a remark in ref. [36]). Both types of intermediate-sized filaments, i.e. those present in densely packed bundles ex- tending throughout the cytoplasm and those contained in the perinuclear whorls ob- served after prolonged drug treatment, al- though they can often be distinguished by their packing densities, have several struc- tural features in common. These include un- stained central cores seen in cross-section (cf PI), “whisker-like” lateral projections (cf [ 10, 12]), and associations with polyribo- somes (cf [ 10, 121).

Cytoskeletal preparations extracted with high salt buffers and Triton X-100 are en- riched in intermediate-sized filaments. Two organizations, i.e. individual filaments of diameters 7-10 nm and laterally aggregated bundles, are recognized in ultrathin sections as well as in negatively stained preparations (fig. 7a, b). The purity and structural pre- servation of the filaments contained in such fractions is comparable to those of similar preparations from other cultured epithelial cells, including rat kangaroo PtK2 and

bovine mammary gland epithelial cells (cf [IO, 17, 291).

Polypeptides enriched in cptoskeletul

preparations

When cytoskeletal preparations obtained by low and high salt buffers and Triton X-100 extraction are examined by SDS-poly- acrylamide gel electrophoresis an enrich- ment of certain specific polypeptide bands is noted (fig. 8). In addition, some of the histones as well as some tightly bound actin is retained (fig. 8). The histones can be re- moved almost completely if treatment with DNAse I following extraction with high salt buffers (procedures (iii) and (iv); see above) is used (fig. 8).

Four polypeptide bands recognized in various cytoskeletal preparations from HeLa cells (fig. 8; slots 3, 5 and 7) appear to be related with polypeptide constituents of intermediate-sized cytoskeletal filaments from other sources. These four polypeptide bands (denoted by arrows in fig. 8, slot 7) have apparent molecular weights of 57000, 52000, 48000, and 46000. The largest of these components (57000 mol. wt) co- migrates with vimentin purified from murine 3T3 cells (fig. 8, slot 4; cf [ 181). The 52000 mol. wt component is the pre- dominant band present in HeLa cytoskele-

Fig. 5. Electron micrographs of ultrathin sections, al- most parallel to the plane of the cell substratum, through HeLa cells grown in monolayer culture. Note the arravs of intermediate-sized filaments in closely packed, bundle-like tibrils, typical for the prekeratin- containing filaments (a, b; normal cells). Sporadically, associations of such bundles with desmosome-like junctions are identified (arrow in b). After treatment of such cells with colcemid (24 h) the extended three- dimensional arrays of bundles of densely packed inter- mediate-sized filaments of the prekeratin-type are maintained (c). HeLa cells contain normal bundles of actin-containing microfilaments (rnf in c) which are especially well developed in the bottom portion of the cell, i.e., the region attached to the substratum. Bars, (a, b) 0.2 pm; (c) 0.5 pm. (a) x70000; (b) X95000; (c) x36ooO.

Keratin in 100 &Zaments of HeLa cells 103

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Fig. 6. Electron micrographs showing the massive quently dense-packed filaments of the prekeratin type perinuclear bundles of intermediate-sized filaments (arrow). The perinuclear intermediate-sized filaments of the vimentin type which can be induced in HeLa are shown, at higher magnification, in (c). NE, nu- cells by prolonged treatment (24 h) with colcemid (a). clear envelope. Bars, (a, b) 0.5 pm; (c) 0.1 pm. (a) (b) Direct comparison of the relatively loosely spaced x40000; (6) x50000; (c) x 100000. filament bundles of the vimentin type and the fre-

tons extracted with high salt buffers and ments of bovine muzzle (slot 2, fig. 8) and Triton X-100 and co-migrates with one of bovine hoof stratum comeum (component the major polypeptides present in prekera- VI; cf [17]). A polypeptide of similar size tin from desmosome-associated tonofila- has also been found in cytoskeleton prep-

Exr, Cell Res 118 (1979)

Keratin in I00 kfilaments of HeLa cells 105

Fig. 7. Enrichment of intermediate-sized filaments in pellets of cytoskeletal preparations from HeLa cells shown in ultrathin sections (a) and in a negatively

arations from various other prekeratin-rich, cultured epithelial cells such as PtK2 and bovine mammary gland epithelium (BMGE; [17, 281). It should also be noted that under our conditions desmin from chicken gizzard and porcine uterus smooth muscle has a similar apparent molecular weight (approx. 53000; fig. 8, slot 1; cf [18]). Polypeptide bands of about 48000 and 45000 apparent molecular weight have also been found in cytoskeletons of BMGE cells and PtK2 cells, respectively [17, 291. As in other cell types [17, 181 comparison of the polypep- tide patterns seen in purified cytoskeletal structures with the polypeptide pattern ob- served in total HeLa cells (fig. 8, slot 6) sug-

stained preparation (uranyl acetate, b). For detailed explanation see text. Bars, (a) 0.5 pm; (b) 0.1 pm. (a) x54000; (b) x 130000.

gests that these four polypeptides together represent a major proportion of the fila- ment proteins of HeLa cells, apparently ex- ceeding the total amount of actin present.

DISCUSSION

We have shown by immunofluorescence microscopy and gel electrophoresis that the majority of intermediate-sized filaments present in growing HeLa cells contain pre- keratin-like proteins and represent a con- siderable fraction of the cytoskeletal pro- teins of these cells. The arrays of the bundles of tonofilament-like intermediate- sized filaments of HeLa cells and their re-

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106 Franke et al.

1234 5 6 7 8 ’

Fig. 8. SDS-polyacrylamide gel electrophoretic separa- tion of polypeptides of material from total HeLa cells (slot 6) and from various cytoskeletal preparations (slots 3, 5 and 7), compared with vimentin purified from mouse 3T3 cytoskeletons (upper band in slot 1 and slot 4), desmin from chicken gizzard (lower band in slot 1), tonotilament-prekeratin from desmosome- tonotilament fractions isolated from bovine muzzle stratum spinosum (slot 2; cf [17]), and various ref- erence proteins (slot 8, from top to bottom: phos- phorylase a, bovine serum albumin, rabbit skeletal muscle actin, chymotrypsinogen). The cytoskeletal material shown in slot 3 has been prepared according to procedure (iii) as described in Methods, that shown in slot 5 has been similarly isolated but extracted twice in high salt buffer, and that shown in slot 7 represents the total TCA-precipitate of the material obtained after treatment with DNAse I according to procedure (ii)

described under Methods. (The band in slot 7 denoted by the arrowhead contains the DNAse 1.) The bands predominant in cytoskeletal preparations enriched in intermediate-sized filaments are indicated by arrows in slot 7 (for apparent molecular weights see text): The uppermost band comigrates with vimentin, and the second band (from top) of apparent molecular weight of 52000 co-migrates with one of the major polypeptides of prekeratin (e.g., slot 2; corresponding to component VI of bovine prekeratin as described in ref. [17]) but is not significantly different in mobility from desmin. (Mammalian desmin has the same electrophoretic mobility on SDS-polyacrylamide gels as chicken desmin, apparent mol. wt 53000, as has been demonstrated by comparison with porcine des- min from uterus smooth muscle; these authors, un- published data.) Note that some actin is retained in cytoskeletons, even after extraction in high salt buffer.

Keratin in 100 A-filaments of HeLa cells 107

arrangement during the mitotic cycle is similar to that described in other cells of epithelial origin [lo, 12, 171. The prekeratin antibodies show strong species cross-reac- tivity (see below), but currently we do not know whether the immunological cross- reaction detects one (perhaps component VI; cf [17]), or more than one, of the pre- keratin polypeptides, and therefore whether these intermediate-sized filaments in HeLa cells contain one or more than one com- ponents in common with prekeratin.

Our results also show that HeLa cells treated with antimitotic drugs contain a sec- ond type of intermediate-sized filaments which cannot be decorated with antibodies to prekeratin. The drug-induced perinuclear aggregates are strongly stained with anti- bodies to murine vimentin (figs 3 b and 4e) although untreated HeLa cells show only a weak and very diffuse staining and no deco- ration of distinct fibrillar structure (fig. 3a). When cytoskeletons from untreated (fig. 8) as well as from colcemid-treated (data not shown here) HeLa cells are examined by SDS-polyacrylamide gel electrophoresis a component migrating with murine vimentin can be observed (fig. 8) but, of course, co- migration does not prove identity. Inter- pretation of these results is complicated by the fact that some antibodies to vimentin show restricted interspecies cross-reac- tivity. However, the antibody to vimentin used in this study appears not to be of this type since it strongly stains the large bundles of intermediate-sized filaments present in human umbilical cord-derived endothelial cells (unpublished results). Currently, therefore, there seem several possible explanations of our ability to stain drug-induced aggregates but not normally occurring vimentin fibers in most cells of, e.g. human, rat kangaroo and chicken ori- gin: (1) The antibodies to vimentin show

only weak reaction in these species and therefore fail to stain individual vimentin fibers but do stain them when aggregated. (2) Vimentin present in drug-induced whorls in HeLa (and other) cells is synthesized only after drug treatment. This explana- tion, however, seems unlikely because the filament whorls are formed in the presence of colcemid during treatment with con- centrations of cycloheximide that effec- tively inhibit protein synthesis (cf [37]). (3) Vimentin is in a different conformation or association in human cells than in most rodent cells. (4) The staining of the peri- nuclear filament whorls is not immune- specific but occurs because of the high con- centration of protein in this region. This, however, seems unlikely since all other sera and purified antibodies used did not stain these structures. Irrespective of which ex- planation is correct the failure to stain the perinuclear aggregates induced by colcemid treatment with the antibodies to prekeratin, which show,no species specificity and react strongly with cells from human, bovine, rodent, rat kangaroo and frog origin argues that the two ‘types of intermediate-sized filaments must be different.

A keratin-like nature of these filaments has been hypothesized in a previous elec- tron microscopic study [2] but the massive accumulations of these filaments in a cer- tain proportion of the cell sample then ex- amined has been regarded as cytopatho- logical phenomenon rather than a basic feature of normal HeLa cell structure. Our present finding of extensive wavy arrays decorated with antibodies to prekeratin in all HeLa cells demonstrates that such fila- ments are a true general element of the HeLa cytoskeleton although their density may differ somewhat under various growth conditions. The specific function of these filaments remains unclear but their general

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108 Franke et al.

cytoskeletal role in the maintenance of the typical cell morphology of epithelial cells can be assumed (cf [10-S, 171). A con- spicuously close relationship of such fila- ments to polyribosomes has also been de- scribed [2, cf 10, 181. It is quite possible that this relationship reflects a specific attach- ment of polyribosomes to such cytoskeletal elements but a fortuitous and/or non-spe- cific association cannot be excluded at the moment.

The electron microscopic observations also suggest that intermediate-sized tila- ments of the vimentin type can be dis- tinguished from those containing prekeratin by their lesser packing degree in the aggre- gate bundles (e.g. fig. 6). Whether or not vimentin and prekeratin polypeptides which form filaments of very similar structure also have underlying basic chemical similarities remains open.

Our observations show that a cell ori- ginally derived from an epithelial tumor [38] classified as a cervical adenocarcinoma [39] which has been maintained in culture since its isolation in 1951 [38, 391 still exhibits characteristic structural features of an epi- thelial cell. These include the synthesis of prekeratin-like proteins and their assembly into tonofilament-like arrays as well as the occasional formation of desmosomal junc- tions. Since immunofluorescence micro- scopy using antibodies against prekeratin- like polypeptides, in combination with anti- bodies against vimentin, seems to be a means to differentiate between epithelia- derived cells and other cells, perhaps in the future antibodies against the different poly- peptides of the various forms of inter- mediate-sized filaments may be of use in the identification of the epithelial or the mesen- chymal origin of a given cell or tumor.

We thank Mrs C. Grund and Mr H. J. Koitzsch for excellent technical assistance. The work has been

partly supported by a grant from the Deutsche Forschungsgemeinschaft (Fr 308/12).

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