TISSUE
& CELL
1979 11 (3) 413-423
Published by Longman Group Ltd. Printed in Great Britain
ROBERT W. RUBIN, JOANNE
HOWARD
and CRAIG
LEONARDI
A BIOCHEMICAL AND ULTRASTRUCTURAL COMPARISON OF TRITON X-100 MODELS NORMAL AND TRANSFORMED CELLS
OF
ABSTRACT. We have compared the two-dimensional gel profiles of Triton models of normal rat kidney (NRK) cells and their Kirsten viral transformant, 442. Several protein differences were detected. The models of the transformed line lacked five acidic oolvoeotides and Dossessed a much higher intermediate filament to actin ratio. Scannina microscopy reveals significant ultra&ctural differences in these models, with td NRK line exhibiting a much more filamentous structure. In addition, nuclease treatment of NRK models causes a dramatic change in their scanning image while the 442 models are unaffected. Nuclease treated models lack microfilaments and appear to contain onlv intermediate filaments, although actin is still a prominent protein constituent. .
that these filaments function asacytoskeleton. In non-Triton extracted tissue cultures, observations have been made comparing microfilament arrangement in normal and transformed cells. By electron microscopy (Altenburg et al., 1976; Goldman et al., 1975; McNutt et al., 1973) or by immunofluorescence staining, (Pollack et al,. 1975) transformed cells contain fewer bundles of microfilaments. The protein nature of the filaments has been studied only superficially by gel electrophoresis. Three major proteins of molecular weights of 42,000,52,000 and 230,OOfl(Brown et al., 1976) have been identified. The first corresponds to actin, the second to intermediate filament protein and the last to LETS protein. However, no systematic protein analysis of these models has been undertaken. The non-ionic detergent cytoskeletal model offers a unique opportunity to compare cells at the structural and biochemical level possessing differing morphology, motility rates and substrate adhesion. Cellular carcinogenesis brings about an alteration in all three of these parameters. Therefore, we investigated the ultrastructure and biochemistry of such models in normal and viral transformed cells. Striking differences
Introduction TISSUE culture cells treated with Triton X-100 or NP-40 lose most of their membranous system (Altenburg et al., 1976; Lehto et al., 1978; Osborn and Weber, 1977; Trotter et al., 1978). The remaining internal structure has been shown by negative staining and indirect immunofluorescence to consist of an extensive network of filaments (Osborn and Weber, 1977; Small and Sofiesyek, 1977; Webster et al., 1978). Two types of filaments were demonstrated, 6-7 nm filaments found in bundles and 10 nm filaments found more randomly scattered (Brown et al., 1976). Lehto et al. (1978) showed in similar models that the 10 nm filaments attached to the nuclear residue. In a similar observation using scanning electron microscopy, 10 and 7 nm filaments were closely associated with the nuclear surface suggesting that they serve to anchor nuclei (Trotter et al., 1978). Further, the original cell morphology is maintained by the filaments after detergent extraction suggesting Department of Anatomy, School of Medicine, P.O. Florida 33101.
University of Miami Box 016960, Miami,
Received 31 January 1979. Revised 20 April 1979. 413
414
RUBIN,
are observed in the protein content and substructure of these models of normal and transformed NRK cells. Materials and Methods Production of cell models
Both the normal rat kidney (NRK) and its Kirsten viral transformant (442) were grown in Dulbecco’s medium with 10% fetal calf serum and 1% penicillin-streptomycin solution. In all cases confluent monolayers were used. Cells were renewed periodically from PPLO free frozen stocks. The cells used for the production of Triton models were handled in a manner similar to that previously described (Brown ef al., 1976) with some important modifications. Media were removed from 75 cm2 Falcon tissue culture flasks. Each flask was washed once with 5 ml of TMGC buffer (136 mM NaCl, 5 mM KCl, 5 mM glucose, 25 mM Tris HCl, 0.5 mM MgCl2, and 0.025 mM CaClo, pH 7.4) (Buckley et al., 1978). pre-warmed to 37°C. Then 2 ml of TMGC buffer plus 0.5% Triton X-100 was added to each flask. The flasks were agitated gently for 5 set and allowed to sit for a period of 5 min. In both normal and transformed cells, the models began to come off the substrate after 2 min. Remaining cells adhering to the flask were removed with a rubber policeman and aspirated vigorously with a Pasteur pipet. The models were removed from the flask, pelleted in a clinical centrifuge at 3000 g for 3 min, and washed twice in TMGC buffer. After each wash, the pellet was resuspended by vigorous vortexing. When viewed in a light microscope, the models from this final pellet retained their original flattened or bipolar shape depending on the cell type. For nuclease experiments, this final cell model pellet was resuspended in 1 ml of TMGC to which was added 20 pg of DNAase 1 (from Bovine pancreas Sigma) and 10 pm of RNAase. Following incubation at 37°C for 1 hr, the preparations were washed in 100 volumes of TMGC, repelleted in a clinical centrifuge and solubilized for one- and twodimensional gel electrophoresis. For scanning or transmission electron microscopy, cells were grown to confluency on carbon-coated cover slips in 2.5 cm Petri dishes. Using Pasteur pipets, a rapid simultaneous exchange of tissue culture media
HOWARD
AND
LEONARD1
with TMGC buffer was performed. This was immediately followed by the exchange of TMGC for TMGC plus Triton X-100. The cells remained in the Triton solution for 2-4 min and then were either exchanged again with TMGC or fixed with TMGC plus Triton and 3% glutaraldehyde buffered in 0.1 M cacodylate, pH 7.4. For the nuclease experiments, models were placed in TMGC plus 20 pm/ml of RNAase and DNAase, respectively, followed by gentle shaking and incubation at 37’ for f-l hr. Prolonged exposure at 37” caused most of the cells to come off the coverslip. Fixation of the nuclease-treated cells was the same as described above. Electron microscopy
Cells were prepared for scanning electron microscopy as described previously (Porter et al., 1972). They were photographed on a JEOL JSM-35 scanning electron microscope. Cells prepared for transmission electron microscopy were grown on carbon-coated cover-slips, treated for model production as described above, and after fixation were embedded in Spurr’s epoxy resin. The coverslips were removed by cold shock and the resulting flat embedded block sectioned with the diamond knife and examined on a Hitachi HU 11C electron microscope. Polyacrylamide
gel analysis
Qualitative and quantitative one-dimensional gels (l-d) were run as previously described (Kahn and Rubin, 1974) were quantitated by cutting out a strip of the slab gel and running this on a Gilford 240 spectrophotometer with the Gilford gel scanning device (Kahn and Rubin, 1974). A modification of the original O’Farrell method (Wilson et al., 1977) was employed for the two-dimensional gel (2-d) analysis. This modification involved the use of SDS in the solubilization procedure, and the use of a larger diameter microcylindrical gel to allow overloading of samples to reveal minor components. Preparations were solubilized within 15 min after the production of the Triton models and were either run the same day or stored at - 9O’C. Extraction of intermediate filament protein
In order to identify the 55,000 dalton component observed on our l-d and 2-d gels,
MODELS
OF
NORMAL
AND
TRANSFORMED
whole Triton models (not nuclease treated) were extracted with 1 M acetic acid. A pellet of cell models from six confluent 75 cm2 Falcon flasks was resuspended in 2 ml of 1 M acetic acid, and dialyzed against 1 liter of 1 M acetic acid on 20 mM NaCl (Small and Sofiesyek, 1977). The resulting suspension was spun at 50,OOOg for 20 min and the supernatant carefully removed. The pH of the supernatant was raised to 3.9, allowed to stand for 15 min at room temperature and spun in the cold for 20 min at 50,OOOg. The resulting pellet was analyzed on l-d gels. Results Production of Triton models We attempted to make cytoskeletal models using 0.5% Triton following the method of Brown et al. (1976). The bulk of the cells detached from the culture flasks during gentle agitation, making it difficult to handle large volumes of cells without excessive loss in the Triton washes. We therefore developed the method described above in which cells are extracted while attached to the substrate and then removed by gentle scraping after the Triton soluble components have been stripped from the cells. The removal of the cells from the substrate surface had no affect on the model morphology at the transmission EM level. Ultrastructure An initial scanning electron microscopic analysis of the Triton models revealed that both lines maintained their previously described shape (Rubin et al., 1978). The structure remaining from the NRK cell line is shown in Fig. 1 (upper left). The cells are flat, closely adhering to the substrate with a flat, centrally located nucleus. The cytoplasmic substructure appears stippled and rough, with little organelle morphology identifiable near the nucleus. The substructure thins into a meshwork at the cell periphery. The 442 Triton models are shown in Fig. 1 (upper right). In these models the nucleus is rounded and the cytoplasmic substructure is formed into hemispherical or cylindrical processes. Near the nucleus the texture is quite rough and pitted. There is the hint of material in bundles stretching from the nucleus into the processes. No recognizable
CELLS
415
substructure that could be correlated to the known actin cables as seen in thin sections of glutaraldehyde fixed whole cells was observed for either cell type. However, the overall cell shape for both NRK and 442 lines (flattened or rounded, respectively) was retained in the Triton model. This is further demonstrated by the retention of the typical telophase morphology in the model (Fig. 1). In thin sections (Fig. 2) no difference was seen between either NRK or 442 models. The models consisted of a nucleus with most of the nucleoplasm present, considerable numbers of ribosomes, and some filaments. The larger randomly oriented filaments had a diameter of 10 nm while fewer filaments were measured at 7 nm. No actin bundles or cables were seen. Since the models were contaminated with nuclear material and ribosomes, we attempted to remove this by prolonged nuclease treatment. In thin section, no difference between the NRK and 442 cells could be detected in nuclease treated models. Most of the ribosomes were removed, exposing the 10 nm filaments. The bulk of the nuclear contents was removed after + hr of nuclease treatment leaving a thin, darkly staining, amorphous material outlining the nucleus. In both cases, actin microfilaments were not seen (Fig. 2). After the application of RNAase and DNAase the substructure of the NRK cell line as seen by scanning EM dramatically changed (Fig. 3). Using either DNAase or RNAase alone produced images similar to the combined treatment of both nucleases. The irregular ill-defined amorphous appearance of the bulk of the cytoplasm was replaced by a network of filaments. These are closely adherent to the substrate and terminate near the cell periphery. The original outline of the cell could still be observed as the termination points of all these filaments. The nucleus itself is either flattened or pushed in and covered by a fine network of filaments. Triton models of the 442 cell line, when treated with nuclease, changed little in morphology. The nucleus appeared collapsed and few filaments were revealed (Fig. 3). Treatment of the models under conditions known to depolymerize actin dialysis against 0.2 mM ATP, 1.0 mM Imidazole-Cl, pH 0.5 (McNutt et al., 1973; Pollack et al., 1973) in other systems neither altered the morphology
MODELS
OF NORMAL
AND
TRANSFORMED
CELLS
Fig. 2. Thin sections of Triton models from 442 cells with (left panel, ~47,000) and without (right panel, x 23,000) nuclease treatment. The lower panel shows the loss of rihosomes, presence of numerous 10 nm filaments organized into a tapering fibril.
Fig. 1. Triton models of NRK (upper left, x 3250) and 442 (upper right, x 13,750) cells. No nuclease treatment was employed. The lower panel (x 1920) is a 442 cell model completing cell division. Some fibrous substructure is apparent at the periphery of this model even though normally absent from interphase 442 cells. 26
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MODELS
OF
NORMAL
AND
TRANSFORMED
CELLS
Fig. 4a, b. Two-dimensional gels of Triton models of NRK (a) and 442 (b) cells. Acid end to the right, low molecular weight toward bottom of gels. Arrow denotes the presence of five spots in the lower right quadrant which are repeatedly present in the NRK and absent in the 442. A number of other differences can be seen but these do not consistently appear when examining numerous preparations. Even though heavily loaded the relative differences in amounts of actin (largest spot in a) and intermediate filament protein (largest spot in b) between the NRK and 442 cell models can be seen. Fig. 4c. Intermediate filament protein and Triton soluble proteins. From left to right: well 1, intermediate filament protein from acetic acid extract of NRK Triton model; well 2, insoluble model; well 3, the Triton soluble proteins which spin down at 50,000 g for 30 min after dialysis against 1 M acetic acid and 20 mM NaCl; the pH of the remaining acidified extract was then raised to 3.9 and then spun at 50,000 g for 20 min; well 4 shows the pellet; well 5, same material as well 4 but with added standard intermediate filament protein; well 6, the remaining supernatant; well 7, same as 6 but with added standard intermediate filament protein. Fig. 4d. From left to right: overloaded one-dimensional slab gels of NRK and 442 Triton models and purified intermediate filament proteins. Intermediate filament protein denoted by the upper arrows, actin by the lower arrows.
Fig. 3. RNAase and DNAase treated NRK (upper left, x 5120; upper right, x 6470) and 442 (lower panel, x 9990) cell Triton models. Note the difference in appearance of the substructure of these two cell types (also compare the lower panel to Fig. 1).
419
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nor released detectable amounts of protein in the supernatant even after a 24-hr incubation. Treatment of models with heavy meromyosin (Lowey and Cohen, 1962; Mommaerts, 1958) by the method of Hinkley and Telser (1974) also failed to alter the thin section image. No decorated filaments of any type were observed under conditions which decorated 5 nm filaments in whole glycerinated cells. Biochemical analysis An extensive l-d and 2-d polyacrylamide gel analysis of the Triton models before and after nuclease treatment revealed two major proteins, actin and intermediate filament protein, which constitute the bulk of the sample. Surprisingly, little change in the overall l-d or 2-d gel patterns were observed after extensive nuclease treatment (not shown). At most, only four to five minor spots were removed by the nuclease treatment followed by extensive washes. It would appear that the bulk of the proteins which constitute the ribosomes and the nuclear contents either represent a small percentage of the total protein in the sample and thus are not resolved, or are solubilized and adhering non-specifically to the remaining cytoskeletal matrix (Fig. 2). The major spot at 55,000 daltons has the same solubility characteristics in acetic acid as intermediate filament protein. As previously reported for smooth muscle, intermediate filament protein (Small and Celis, 1978) is soluble in 1 M acetic acid in the presence of 20 mM NaCI. When NRK or 442 Triton models were acid extracted, and the extract precipitated at pH 3.9 (Small and Sofiesyek, 1977), the resulting pellet contained over 95% of the 55,000 dalton molecular weight protein (Fig. 5). It was also found that the intermediate filament protein has an isoelectric point and molecular weight which in our system was identical to the alpha subunit of bovine brain tubulin. This was determined by coelectrophoresis on 2-d gels with purified bovine brain tubulin under a variety of protein concentrations. This may be due to the gel system employed, for others (Lazarides and Hubbard, 1976) have been able to resolve a 55,000 dalton band from tubulin on SDS gels. An extensive study of the NRK and 442
RUBIN,
HOWARD
AND LEONARD1
cell models without nuclease treatment was then undertaken. They were compared and all of the minor spots were catalogued. In addition to the actin and intermediate filament protein, over 37 individual components could repeatedly be resolved (see Fig. 4). Two dramatic and repeatable differences in the protein content of the NRK and the 442 models are readily apparent. First, the ratio of the actin to the intermediate filament protein was different, as determined by the method of Kahn and Rubin (1974). For the NRK model, the ratio (Goldman et a/., 1975) of actin to the 55,000 mol. wt protein was I : 1.2. For the 442 models, this ratio was I :5.3. This actin represents 446% of the total cellular protein No tubulin was present in these models as no tubulin spots could be detected on 2-d gels. At confluency, both cell lines contain the same amount of total actin (Rubin et a/., 1978). The second difference is the absence of five spots in the acid end of the pH gradient in the 442 cell line Triton models (Fig. 4). Numerous other minor differences in the spot patterns were also frequently observed (Fig. 4). However, only these five spots were consistently different in all experiments. The Triton soluble fraction was investigated in the NRK and 442 cells. As no 55,000 mol. wt protein was detectable in these preparations (Fig. 4), we conclude that either no pool exists or that it is present at a small fraction of 1% of the total intermediate filament protein content. In order to test the possibility that the protein differences observed between the NRK and 442 cells were caused by nuclear contamination, nuclei were isolated (Lenk et al., 1977) and run on 2-d gels. A small amount of actin and 55,000 mol. wt protein ran into the gel but no other spots were resolved. These two spots probably represent contamination from the cytoskeleton as some remaining filaments were observed in section material. However, even when the gels were overloaded, the results indicated little or no other proteins entering or remaining in the first dimensional gel, Discussion Our results indicate that Triton models of normal NRK cells differ structurally and biochemically from their Kristen viral trans-
MODELS
OF
NORMAL
AND
TRANSFORMED
formant. Nuclease-treated models of NRK show a fibrous substructure which is absent in nuclease-treated 442 cells. These NRK models also possess a higher ratio of actin to intermediate filament protein than in the 442 models. Lastly, the NRK models possess five unique polypeptides which are absent in the 442 models. The structural difference, although not revealed at the thin section level, is observed in the scanning image. The difference in the scanning image between NRK and 442 models is most dramatically demonstrated in the nuclease-treated models. NRK models have a spongy network of filaments that is comparable to those observed by Trotter et al. (1978) in 3T3 cells. The latter cells were not treated with a nuclease but were treated with a more concentrated solution of Triton. The overall topography of the nuclease-treated Triton models is different from the living cell in that in viva, the NRK line has numerous actin cables or bundles running through the base of the cytoplasm and outlining the shape of the cell. Actin bundles outlining the periphery of the cell were not clearly demonstrated in either the untreated or nuclease-treated cells. However, filaments were seen extending from the nucleus in the NRK models in both thin sections and scanning EM. They are comparable to the intermediate filaments seen by Trotter et al. (1978). Nuclease-treated models are seen after conventional transmission electron microscopic preparation to contain no microfilament bundles, even though Brown et al. (1976) and others (Pollack et al., 1975; Trotter et al., 1978), using negative staining and immunofluorescent techniques, were able to resolve numerous actin cables in similar models of fibroblast cells. We may assume that osmication or some other step in EM preparation has depolymerized the F-actin (Maupin-Szamier and Pollard, 1978). An alternative assumption is that these epithelial-like cell lines possess a much less well-developed actin bundle system than fibroblast cells. Untreated models do contain a few 50-70 A free filaments as seen in thin sections. These are apparently depolymerized by the DNAase treatment. Nuclease treatment apparently reduces or eliminates the material between these bundles. This suggests that polyribosome cytoskeletal elements (presumable mRNA) reported previously (Lenk, et al., 1977) serve an important
CELLS
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cytoskeletal function. The DNAase sensitive component (probably F-actin) may also serve to maintain lateral cytoskeletal connections. The lack of demonstration of protein removal using 2-d gels after RNAase and DNAase treatment implies that the nuclear and ribosomal proteins which remain after Triton extraction do not enter the isoelectric focusing gel or run off the gel entirely and are thus not resolved in our 2-d gel system. Since many of these proteins have a high negative charge, this is not a surprising result and is supported by our gels of isolated nuclei. Therefore. we feel that the bulk of the proteins observed in our 2-d gels do represent cytoskeletal elements. Another point relating to the utility of the Triton model system is the observation that the model actin represents 46% of the total cell protein. In a previous observation, we reported using different methods (Rubin et al., 1978), that NRK cells possess a maximum of no more than 50% of their total actin in a pelletable form and that all cellular actin equals no more than 10 o/0of the total protein. Thus the Triton models appear to contain all of the cells’ pelletable actin. Considerable soluble actin is extracted in the Triton supernatant. The complete absence of the 55,000 dalton protein in the extracted Triton supernatants as revealed by both one- and two-dimensional gel electrophoresis supports the recent suggestion (Hynes and Destrel, 1978) that all intermediate filament protein is polymerized in the cell. Consistent differences in the protein composition of the normal and transformed cell models with and without nuclease treatment does suggest that these proteins may play a role in the production of the ultrastructural differences between these two cells and their cell models. On the basis of the differential effects of colchicine on 10 nm filaments in normal and transformed cells, Hynes and Destrel (1978) have suggested that 10 nm filaments become altered in their interaction with other cellular components upon transformation. A comparison of the protein composition from the cytoskeleton of these lines could be used to deal with the problem of whether the changes we have observed relate to the transformation process and tumorigenicity or whether it is something that relates solely
RUBIN,
422
to the morphological differences between our two cell lines. A protein analysis of Triton models of normal and transformed hamster fibroblast cell lines was recently reported to demonstrate a great decrease in a 55,000 dalton protein in the transformed line (Tuszynski et aI., 1978). Apparently, this protein is different from our 55 K protein as revealed by 2-d gel analysis. Lastly, nuclease treatment of the Triton models (either NRK or 442), although not dramatically changing the two-dimensional gel pattern, does eliminate all microfilamentous material in thin section from these models. Perhaps the DNAase is depolymerizing some of the F-actin since DNAase is a strong and specific actin binding protein. We have not yet quantitated the possible release of actin into the supernatant after such treatment, but it is clear that a great deal of actin is still retained. Roughly 30% of the total cell protein of the model after nuclease treatment is actin. The
HOWARD
AND
LEONARD1
nuclease treated models in thin section appear to contain only intermediate filaments. We therefore suggest that intermediate filaments contain at least two proteins, intermediate filament protein of molecular weight 55,000 daltons and actin. In addition, after mild protease treatment, intermediate filaments have been shown to bind heavy meromyosin (Buckley et al., 1978). Alternatively, the nuclease treated models might be particularly sensitive to osmium induced actin depolymerization (Maupin-Szamier and Pollard, 1978). It is hoped that further examination of non-ionic detergent models of normal and transformed cells can reveal the ultimate protein alterations that result in altered cellular motility and morphology during cellular carcinogenesis. Acknowledgements
This work was supported No. 1 ROl CA 19855 02.
by NC1 Grant
References ALTENBURG, B. C. ,SOMMERS, K. and STEINER,S. 1976. Altered microfilament structure in cells transformed with a temperature-sensitive transformation mutant of murine sarcoma virus. Cancer Rrs., 36, 251-257. BROWN, S., LEVENSON, W. and SPUDICH, J. A. 1976. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J. Supramol. Struct., 5, 119-130. BUCKLEY, I. K., RAJA, T. R. and STEWART, M. 1978. Heavy meromyosin labeling of intermediate filaments in cultured connective tissue cells. J. Cell Biol., 78, 644-652. GOLDMAN, R. D., CHANG, C. and WILLIAMS, J. F. 1975. Properties and behavior of hamster embryo cells transformed by human adenovirus type 5. Cold Spring Hub. Symp. quant. Biol., 37, 523-533. HINKLEY, R. and TELSER, A. 1974. Heavy meromyosin-binding filaments in the mitotic apparatus of mammalian cells. Exp. Cell Res., 86, 161-164. HYNES, R. 0. and DESTREL, A. T. 1978. 10 nm filaments in normal and transformed cells. Cell, 13, 151-163. KAHN, R., and RUBIN, R. W. 1974. Quantitation ofprotein on slab SDS polyacryiamide gels using Coomassie Blue. Amlyt. Biochem., 67, 347-352. LAZARIDES, E. and HUBBARD, B. D. 1976. Immunological characterization of the subunit of the 100 A filaments from muscle cells. Proc. Nor. Acad. Sci., 73, 43444348. LEHTO, V., VIRTANEN,1. and PEKKA, K. 1978. Intermediate filaments anchor the nuclei in nuclear monolayers of cultured human fibroblasts. Nature, Lond., 272, 175-I 77. LENK, R., RANSOM, L., KAUFMANN, Y. and PENMAN, S. 1977. A cytoskeletal structure with associated polyribosomes from HeLa cells. Cell, 10, 67-78. LOWEY, S. and COHEN, C. 1962. Studies on the structure of myosin. J. Mol. Biol., 4, 293-308. MAUPIN-SZAMIER, P. and POLLARD, T. D. 1978. Actin filament destruction by osmium tetroxide. J. Cc>/1 Biol. 17, 837-852. MOMMAERTS,W. F. 1958. Chemical investigations of muscular tissue. Mrthod.v of’ Med. Res., (ed. J. V. Warren), Vol. 7, p. 3. Yearbook Medical Publishers, Chicago. MCNUTT, N. S., CULP, L. A. and BLACK, P. H. 1973. Contact-inhibited revertant cell lines isolated from SV&ransformed cells. IV. Microfilament distribution and cell shape in untransformed, transformed and revertant balb 3T3 cells. J. Cell Biof., 56, 412428. OSBORN, M. and WEBER, K. 1977. The detergent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Expl. CeN Res., 106, 339-349.
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POLLACK, R., OSBORN, M. and WEBER, K. 1975. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc. Nat. Acad. Sri. U.S.A., 12, 994-998. POLLARD, T. D. and KORN, E. D. 1973. Electron microscopic identification of actin associated with isolated amoeba plasma membranes. J. biol. Chem., 248, 448-450. PORTER, K. R., KELLEY, D. and ANDREWS, P. M. 1972. Proc. Fifth Annual Stereoscan Colloquium. RUBIN, R. W., WARREN, R. H., LUKEMAN, D. S. and CLEMENTS,E. 1978. Actin content and organization in normal and transformed cells in culture. J. Cell Viol., 78, 28. SMALL, J. V. and CELIS, J. E. 1978. Direct visualization of the 10 nm (100 A) filament network in whole and enucleated culture cells. J. Cell Sci., 31, 393409. SMALL, J. V. and SOFIESYEK,A. 1977. Studies on the function and comparison of the 10 nm (100 A) filaments of vertebrate smooth muscle. J. Cc/f Sci., 23, 243-268. TROTTER, J. A., FOERDER, B. A. and KELLER, J. M. 1978. Intracellular fibersin cultured cells: analysis by scanning and transmission electron microscopy and by SDS-polyacrylamide gel electrophoresis. J. CeN Sri., 31, 369-392. TUSZYNSKI, G. P., FRANK, E. and DANSKY, C. 1978. Cold Spring Harbor Symp. Cytoskeleton and Cell Motility. WEBSTER, R. E., HENDERSON,D., OSBORN, M. and WEBER, K. 1978. Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin and tubulin containing structures. Proc. Nat. Acad. Sci., 75, 5511-5515. WILSON, D. L., HALL, M. E., STONE, G. C. and RUBIN, R. W. 1977. Some improvements in two-dimensional gel electrophoresis of proteins: protein mapping of eukaryotic tissue extracts. Analyt. Eiochem., 83, 33-44.
& CELL
1979 11 (3) 413-423
Published by Longman Group Ltd. Printed in Great Britain
ROBERT W. RUBIN, JOANNE
HOWARD
and CRAIG
LEONARDI
A BIOCHEMICAL AND ULTRASTRUCTURAL COMPARISON OF TRITON X-100 MODELS NORMAL AND TRANSFORMED CELLS
OF
ABSTRACT. We have compared the two-dimensional gel profiles of Triton models of normal rat kidney (NRK) cells and their Kirsten viral transformant, 442. Several protein differences were detected. The models of the transformed line lacked five acidic oolvoeotides and Dossessed a much higher intermediate filament to actin ratio. Scannina microscopy reveals significant ultra&ctural differences in these models, with td NRK line exhibiting a much more filamentous structure. In addition, nuclease treatment of NRK models causes a dramatic change in their scanning image while the 442 models are unaffected. Nuclease treated models lack microfilaments and appear to contain onlv intermediate filaments, although actin is still a prominent protein constituent. .
that these filaments function asacytoskeleton. In non-Triton extracted tissue cultures, observations have been made comparing microfilament arrangement in normal and transformed cells. By electron microscopy (Altenburg et al., 1976; Goldman et al., 1975; McNutt et al., 1973) or by immunofluorescence staining, (Pollack et al,. 1975) transformed cells contain fewer bundles of microfilaments. The protein nature of the filaments has been studied only superficially by gel electrophoresis. Three major proteins of molecular weights of 42,000,52,000 and 230,OOfl(Brown et al., 1976) have been identified. The first corresponds to actin, the second to intermediate filament protein and the last to LETS protein. However, no systematic protein analysis of these models has been undertaken. The non-ionic detergent cytoskeletal model offers a unique opportunity to compare cells at the structural and biochemical level possessing differing morphology, motility rates and substrate adhesion. Cellular carcinogenesis brings about an alteration in all three of these parameters. Therefore, we investigated the ultrastructure and biochemistry of such models in normal and viral transformed cells. Striking differences
Introduction TISSUE culture cells treated with Triton X-100 or NP-40 lose most of their membranous system (Altenburg et al., 1976; Lehto et al., 1978; Osborn and Weber, 1977; Trotter et al., 1978). The remaining internal structure has been shown by negative staining and indirect immunofluorescence to consist of an extensive network of filaments (Osborn and Weber, 1977; Small and Sofiesyek, 1977; Webster et al., 1978). Two types of filaments were demonstrated, 6-7 nm filaments found in bundles and 10 nm filaments found more randomly scattered (Brown et al., 1976). Lehto et al. (1978) showed in similar models that the 10 nm filaments attached to the nuclear residue. In a similar observation using scanning electron microscopy, 10 and 7 nm filaments were closely associated with the nuclear surface suggesting that they serve to anchor nuclei (Trotter et al., 1978). Further, the original cell morphology is maintained by the filaments after detergent extraction suggesting Department of Anatomy, School of Medicine, P.O. Florida 33101.
University of Miami Box 016960, Miami,
Received 31 January 1979. Revised 20 April 1979. 413
414
RUBIN,
are observed in the protein content and substructure of these models of normal and transformed NRK cells. Materials and Methods Production of cell models
Both the normal rat kidney (NRK) and its Kirsten viral transformant (442) were grown in Dulbecco’s medium with 10% fetal calf serum and 1% penicillin-streptomycin solution. In all cases confluent monolayers were used. Cells were renewed periodically from PPLO free frozen stocks. The cells used for the production of Triton models were handled in a manner similar to that previously described (Brown ef al., 1976) with some important modifications. Media were removed from 75 cm2 Falcon tissue culture flasks. Each flask was washed once with 5 ml of TMGC buffer (136 mM NaCl, 5 mM KCl, 5 mM glucose, 25 mM Tris HCl, 0.5 mM MgCl2, and 0.025 mM CaClo, pH 7.4) (Buckley et al., 1978). pre-warmed to 37°C. Then 2 ml of TMGC buffer plus 0.5% Triton X-100 was added to each flask. The flasks were agitated gently for 5 set and allowed to sit for a period of 5 min. In both normal and transformed cells, the models began to come off the substrate after 2 min. Remaining cells adhering to the flask were removed with a rubber policeman and aspirated vigorously with a Pasteur pipet. The models were removed from the flask, pelleted in a clinical centrifuge at 3000 g for 3 min, and washed twice in TMGC buffer. After each wash, the pellet was resuspended by vigorous vortexing. When viewed in a light microscope, the models from this final pellet retained their original flattened or bipolar shape depending on the cell type. For nuclease experiments, this final cell model pellet was resuspended in 1 ml of TMGC to which was added 20 pg of DNAase 1 (from Bovine pancreas Sigma) and 10 pm of RNAase. Following incubation at 37°C for 1 hr, the preparations were washed in 100 volumes of TMGC, repelleted in a clinical centrifuge and solubilized for one- and twodimensional gel electrophoresis. For scanning or transmission electron microscopy, cells were grown to confluency on carbon-coated cover slips in 2.5 cm Petri dishes. Using Pasteur pipets, a rapid simultaneous exchange of tissue culture media
HOWARD
AND
LEONARD1
with TMGC buffer was performed. This was immediately followed by the exchange of TMGC for TMGC plus Triton X-100. The cells remained in the Triton solution for 2-4 min and then were either exchanged again with TMGC or fixed with TMGC plus Triton and 3% glutaraldehyde buffered in 0.1 M cacodylate, pH 7.4. For the nuclease experiments, models were placed in TMGC plus 20 pm/ml of RNAase and DNAase, respectively, followed by gentle shaking and incubation at 37’ for f-l hr. Prolonged exposure at 37” caused most of the cells to come off the coverslip. Fixation of the nuclease-treated cells was the same as described above. Electron microscopy
Cells were prepared for scanning electron microscopy as described previously (Porter et al., 1972). They were photographed on a JEOL JSM-35 scanning electron microscope. Cells prepared for transmission electron microscopy were grown on carbon-coated cover-slips, treated for model production as described above, and after fixation were embedded in Spurr’s epoxy resin. The coverslips were removed by cold shock and the resulting flat embedded block sectioned with the diamond knife and examined on a Hitachi HU 11C electron microscope. Polyacrylamide
gel analysis
Qualitative and quantitative one-dimensional gels (l-d) were run as previously described (Kahn and Rubin, 1974) were quantitated by cutting out a strip of the slab gel and running this on a Gilford 240 spectrophotometer with the Gilford gel scanning device (Kahn and Rubin, 1974). A modification of the original O’Farrell method (Wilson et al., 1977) was employed for the two-dimensional gel (2-d) analysis. This modification involved the use of SDS in the solubilization procedure, and the use of a larger diameter microcylindrical gel to allow overloading of samples to reveal minor components. Preparations were solubilized within 15 min after the production of the Triton models and were either run the same day or stored at - 9O’C. Extraction of intermediate filament protein
In order to identify the 55,000 dalton component observed on our l-d and 2-d gels,
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whole Triton models (not nuclease treated) were extracted with 1 M acetic acid. A pellet of cell models from six confluent 75 cm2 Falcon flasks was resuspended in 2 ml of 1 M acetic acid, and dialyzed against 1 liter of 1 M acetic acid on 20 mM NaCl (Small and Sofiesyek, 1977). The resulting suspension was spun at 50,OOOg for 20 min and the supernatant carefully removed. The pH of the supernatant was raised to 3.9, allowed to stand for 15 min at room temperature and spun in the cold for 20 min at 50,OOOg. The resulting pellet was analyzed on l-d gels. Results Production of Triton models We attempted to make cytoskeletal models using 0.5% Triton following the method of Brown et al. (1976). The bulk of the cells detached from the culture flasks during gentle agitation, making it difficult to handle large volumes of cells without excessive loss in the Triton washes. We therefore developed the method described above in which cells are extracted while attached to the substrate and then removed by gentle scraping after the Triton soluble components have been stripped from the cells. The removal of the cells from the substrate surface had no affect on the model morphology at the transmission EM level. Ultrastructure An initial scanning electron microscopic analysis of the Triton models revealed that both lines maintained their previously described shape (Rubin et al., 1978). The structure remaining from the NRK cell line is shown in Fig. 1 (upper left). The cells are flat, closely adhering to the substrate with a flat, centrally located nucleus. The cytoplasmic substructure appears stippled and rough, with little organelle morphology identifiable near the nucleus. The substructure thins into a meshwork at the cell periphery. The 442 Triton models are shown in Fig. 1 (upper right). In these models the nucleus is rounded and the cytoplasmic substructure is formed into hemispherical or cylindrical processes. Near the nucleus the texture is quite rough and pitted. There is the hint of material in bundles stretching from the nucleus into the processes. No recognizable
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substructure that could be correlated to the known actin cables as seen in thin sections of glutaraldehyde fixed whole cells was observed for either cell type. However, the overall cell shape for both NRK and 442 lines (flattened or rounded, respectively) was retained in the Triton model. This is further demonstrated by the retention of the typical telophase morphology in the model (Fig. 1). In thin sections (Fig. 2) no difference was seen between either NRK or 442 models. The models consisted of a nucleus with most of the nucleoplasm present, considerable numbers of ribosomes, and some filaments. The larger randomly oriented filaments had a diameter of 10 nm while fewer filaments were measured at 7 nm. No actin bundles or cables were seen. Since the models were contaminated with nuclear material and ribosomes, we attempted to remove this by prolonged nuclease treatment. In thin section, no difference between the NRK and 442 cells could be detected in nuclease treated models. Most of the ribosomes were removed, exposing the 10 nm filaments. The bulk of the nuclear contents was removed after + hr of nuclease treatment leaving a thin, darkly staining, amorphous material outlining the nucleus. In both cases, actin microfilaments were not seen (Fig. 2). After the application of RNAase and DNAase the substructure of the NRK cell line as seen by scanning EM dramatically changed (Fig. 3). Using either DNAase or RNAase alone produced images similar to the combined treatment of both nucleases. The irregular ill-defined amorphous appearance of the bulk of the cytoplasm was replaced by a network of filaments. These are closely adherent to the substrate and terminate near the cell periphery. The original outline of the cell could still be observed as the termination points of all these filaments. The nucleus itself is either flattened or pushed in and covered by a fine network of filaments. Triton models of the 442 cell line, when treated with nuclease, changed little in morphology. The nucleus appeared collapsed and few filaments were revealed (Fig. 3). Treatment of the models under conditions known to depolymerize actin dialysis against 0.2 mM ATP, 1.0 mM Imidazole-Cl, pH 0.5 (McNutt et al., 1973; Pollack et al., 1973) in other systems neither altered the morphology
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Fig. 2. Thin sections of Triton models from 442 cells with (left panel, ~47,000) and without (right panel, x 23,000) nuclease treatment. The lower panel shows the loss of rihosomes, presence of numerous 10 nm filaments organized into a tapering fibril.
Fig. 1. Triton models of NRK (upper left, x 3250) and 442 (upper right, x 13,750) cells. No nuclease treatment was employed. The lower panel (x 1920) is a 442 cell model completing cell division. Some fibrous substructure is apparent at the periphery of this model even though normally absent from interphase 442 cells. 26
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Fig. 4a, b. Two-dimensional gels of Triton models of NRK (a) and 442 (b) cells. Acid end to the right, low molecular weight toward bottom of gels. Arrow denotes the presence of five spots in the lower right quadrant which are repeatedly present in the NRK and absent in the 442. A number of other differences can be seen but these do not consistently appear when examining numerous preparations. Even though heavily loaded the relative differences in amounts of actin (largest spot in a) and intermediate filament protein (largest spot in b) between the NRK and 442 cell models can be seen. Fig. 4c. Intermediate filament protein and Triton soluble proteins. From left to right: well 1, intermediate filament protein from acetic acid extract of NRK Triton model; well 2, insoluble model; well 3, the Triton soluble proteins which spin down at 50,000 g for 30 min after dialysis against 1 M acetic acid and 20 mM NaCl; the pH of the remaining acidified extract was then raised to 3.9 and then spun at 50,000 g for 20 min; well 4 shows the pellet; well 5, same material as well 4 but with added standard intermediate filament protein; well 6, the remaining supernatant; well 7, same as 6 but with added standard intermediate filament protein. Fig. 4d. From left to right: overloaded one-dimensional slab gels of NRK and 442 Triton models and purified intermediate filament proteins. Intermediate filament protein denoted by the upper arrows, actin by the lower arrows.
Fig. 3. RNAase and DNAase treated NRK (upper left, x 5120; upper right, x 6470) and 442 (lower panel, x 9990) cell Triton models. Note the difference in appearance of the substructure of these two cell types (also compare the lower panel to Fig. 1).
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nor released detectable amounts of protein in the supernatant even after a 24-hr incubation. Treatment of models with heavy meromyosin (Lowey and Cohen, 1962; Mommaerts, 1958) by the method of Hinkley and Telser (1974) also failed to alter the thin section image. No decorated filaments of any type were observed under conditions which decorated 5 nm filaments in whole glycerinated cells. Biochemical analysis An extensive l-d and 2-d polyacrylamide gel analysis of the Triton models before and after nuclease treatment revealed two major proteins, actin and intermediate filament protein, which constitute the bulk of the sample. Surprisingly, little change in the overall l-d or 2-d gel patterns were observed after extensive nuclease treatment (not shown). At most, only four to five minor spots were removed by the nuclease treatment followed by extensive washes. It would appear that the bulk of the proteins which constitute the ribosomes and the nuclear contents either represent a small percentage of the total protein in the sample and thus are not resolved, or are solubilized and adhering non-specifically to the remaining cytoskeletal matrix (Fig. 2). The major spot at 55,000 daltons has the same solubility characteristics in acetic acid as intermediate filament protein. As previously reported for smooth muscle, intermediate filament protein (Small and Celis, 1978) is soluble in 1 M acetic acid in the presence of 20 mM NaCI. When NRK or 442 Triton models were acid extracted, and the extract precipitated at pH 3.9 (Small and Sofiesyek, 1977), the resulting pellet contained over 95% of the 55,000 dalton molecular weight protein (Fig. 5). It was also found that the intermediate filament protein has an isoelectric point and molecular weight which in our system was identical to the alpha subunit of bovine brain tubulin. This was determined by coelectrophoresis on 2-d gels with purified bovine brain tubulin under a variety of protein concentrations. This may be due to the gel system employed, for others (Lazarides and Hubbard, 1976) have been able to resolve a 55,000 dalton band from tubulin on SDS gels. An extensive study of the NRK and 442
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cell models without nuclease treatment was then undertaken. They were compared and all of the minor spots were catalogued. In addition to the actin and intermediate filament protein, over 37 individual components could repeatedly be resolved (see Fig. 4). Two dramatic and repeatable differences in the protein content of the NRK and the 442 models are readily apparent. First, the ratio of the actin to the intermediate filament protein was different, as determined by the method of Kahn and Rubin (1974). For the NRK model, the ratio (Goldman et a/., 1975) of actin to the 55,000 mol. wt protein was I : 1.2. For the 442 models, this ratio was I :5.3. This actin represents 446% of the total cellular protein No tubulin was present in these models as no tubulin spots could be detected on 2-d gels. At confluency, both cell lines contain the same amount of total actin (Rubin et a/., 1978). The second difference is the absence of five spots in the acid end of the pH gradient in the 442 cell line Triton models (Fig. 4). Numerous other minor differences in the spot patterns were also frequently observed (Fig. 4). However, only these five spots were consistently different in all experiments. The Triton soluble fraction was investigated in the NRK and 442 cells. As no 55,000 mol. wt protein was detectable in these preparations (Fig. 4), we conclude that either no pool exists or that it is present at a small fraction of 1% of the total intermediate filament protein content. In order to test the possibility that the protein differences observed between the NRK and 442 cells were caused by nuclear contamination, nuclei were isolated (Lenk et al., 1977) and run on 2-d gels. A small amount of actin and 55,000 mol. wt protein ran into the gel but no other spots were resolved. These two spots probably represent contamination from the cytoskeleton as some remaining filaments were observed in section material. However, even when the gels were overloaded, the results indicated little or no other proteins entering or remaining in the first dimensional gel, Discussion Our results indicate that Triton models of normal NRK cells differ structurally and biochemically from their Kristen viral trans-
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formant. Nuclease-treated models of NRK show a fibrous substructure which is absent in nuclease-treated 442 cells. These NRK models also possess a higher ratio of actin to intermediate filament protein than in the 442 models. Lastly, the NRK models possess five unique polypeptides which are absent in the 442 models. The structural difference, although not revealed at the thin section level, is observed in the scanning image. The difference in the scanning image between NRK and 442 models is most dramatically demonstrated in the nuclease-treated models. NRK models have a spongy network of filaments that is comparable to those observed by Trotter et al. (1978) in 3T3 cells. The latter cells were not treated with a nuclease but were treated with a more concentrated solution of Triton. The overall topography of the nuclease-treated Triton models is different from the living cell in that in viva, the NRK line has numerous actin cables or bundles running through the base of the cytoplasm and outlining the shape of the cell. Actin bundles outlining the periphery of the cell were not clearly demonstrated in either the untreated or nuclease-treated cells. However, filaments were seen extending from the nucleus in the NRK models in both thin sections and scanning EM. They are comparable to the intermediate filaments seen by Trotter et al. (1978). Nuclease-treated models are seen after conventional transmission electron microscopic preparation to contain no microfilament bundles, even though Brown et al. (1976) and others (Pollack et al., 1975; Trotter et al., 1978), using negative staining and immunofluorescent techniques, were able to resolve numerous actin cables in similar models of fibroblast cells. We may assume that osmication or some other step in EM preparation has depolymerized the F-actin (Maupin-Szamier and Pollard, 1978). An alternative assumption is that these epithelial-like cell lines possess a much less well-developed actin bundle system than fibroblast cells. Untreated models do contain a few 50-70 A free filaments as seen in thin sections. These are apparently depolymerized by the DNAase treatment. Nuclease treatment apparently reduces or eliminates the material between these bundles. This suggests that polyribosome cytoskeletal elements (presumable mRNA) reported previously (Lenk, et al., 1977) serve an important
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cytoskeletal function. The DNAase sensitive component (probably F-actin) may also serve to maintain lateral cytoskeletal connections. The lack of demonstration of protein removal using 2-d gels after RNAase and DNAase treatment implies that the nuclear and ribosomal proteins which remain after Triton extraction do not enter the isoelectric focusing gel or run off the gel entirely and are thus not resolved in our 2-d gel system. Since many of these proteins have a high negative charge, this is not a surprising result and is supported by our gels of isolated nuclei. Therefore. we feel that the bulk of the proteins observed in our 2-d gels do represent cytoskeletal elements. Another point relating to the utility of the Triton model system is the observation that the model actin represents 46% of the total cell protein. In a previous observation, we reported using different methods (Rubin et al., 1978), that NRK cells possess a maximum of no more than 50% of their total actin in a pelletable form and that all cellular actin equals no more than 10 o/0of the total protein. Thus the Triton models appear to contain all of the cells’ pelletable actin. Considerable soluble actin is extracted in the Triton supernatant. The complete absence of the 55,000 dalton protein in the extracted Triton supernatants as revealed by both one- and two-dimensional gel electrophoresis supports the recent suggestion (Hynes and Destrel, 1978) that all intermediate filament protein is polymerized in the cell. Consistent differences in the protein composition of the normal and transformed cell models with and without nuclease treatment does suggest that these proteins may play a role in the production of the ultrastructural differences between these two cells and their cell models. On the basis of the differential effects of colchicine on 10 nm filaments in normal and transformed cells, Hynes and Destrel (1978) have suggested that 10 nm filaments become altered in their interaction with other cellular components upon transformation. A comparison of the protein composition from the cytoskeleton of these lines could be used to deal with the problem of whether the changes we have observed relate to the transformation process and tumorigenicity or whether it is something that relates solely
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to the morphological differences between our two cell lines. A protein analysis of Triton models of normal and transformed hamster fibroblast cell lines was recently reported to demonstrate a great decrease in a 55,000 dalton protein in the transformed line (Tuszynski et aI., 1978). Apparently, this protein is different from our 55 K protein as revealed by 2-d gel analysis. Lastly, nuclease treatment of the Triton models (either NRK or 442), although not dramatically changing the two-dimensional gel pattern, does eliminate all microfilamentous material in thin section from these models. Perhaps the DNAase is depolymerizing some of the F-actin since DNAase is a strong and specific actin binding protein. We have not yet quantitated the possible release of actin into the supernatant after such treatment, but it is clear that a great deal of actin is still retained. Roughly 30% of the total cell protein of the model after nuclease treatment is actin. The
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nuclease treated models in thin section appear to contain only intermediate filaments. We therefore suggest that intermediate filaments contain at least two proteins, intermediate filament protein of molecular weight 55,000 daltons and actin. In addition, after mild protease treatment, intermediate filaments have been shown to bind heavy meromyosin (Buckley et al., 1978). Alternatively, the nuclease treated models might be particularly sensitive to osmium induced actin depolymerization (Maupin-Szamier and Pollard, 1978). It is hoped that further examination of non-ionic detergent models of normal and transformed cells can reveal the ultimate protein alterations that result in altered cellular motility and morphology during cellular carcinogenesis. Acknowledgements
This work was supported No. 1 ROl CA 19855 02.
by NC1 Grant
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POLLACK, R., OSBORN, M. and WEBER, K. 1975. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc. Nat. Acad. Sri. U.S.A., 12, 994-998. POLLARD, T. D. and KORN, E. D. 1973. Electron microscopic identification of actin associated with isolated amoeba plasma membranes. J. biol. Chem., 248, 448-450. PORTER, K. R., KELLEY, D. and ANDREWS, P. M. 1972. Proc. Fifth Annual Stereoscan Colloquium. RUBIN, R. W., WARREN, R. H., LUKEMAN, D. S. and CLEMENTS,E. 1978. Actin content and organization in normal and transformed cells in culture. J. Cell Viol., 78, 28. SMALL, J. V. and CELIS, J. E. 1978. Direct visualization of the 10 nm (100 A) filament network in whole and enucleated culture cells. J. Cell Sci., 31, 393409. SMALL, J. V. and SOFIESYEK,A. 1977. Studies on the function and comparison of the 10 nm (100 A) filaments of vertebrate smooth muscle. J. Cc/f Sci., 23, 243-268. TROTTER, J. A., FOERDER, B. A. and KELLER, J. M. 1978. Intracellular fibersin cultured cells: analysis by scanning and transmission electron microscopy and by SDS-polyacrylamide gel electrophoresis. J. CeN Sri., 31, 369-392. TUSZYNSKI, G. P., FRANK, E. and DANSKY, C. 1978. Cold Spring Harbor Symp. Cytoskeleton and Cell Motility. WEBSTER, R. E., HENDERSON,D., OSBORN, M. and WEBER, K. 1978. Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin and tubulin containing structures. Proc. Nat. Acad. Sci., 75, 5511-5515. WILSON, D. L., HALL, M. E., STONE, G. C. and RUBIN, R. W. 1977. Some improvements in two-dimensional gel electrophoresis of proteins: protein mapping of eukaryotic tissue extracts. Analyt. Eiochem., 83, 33-44.
