Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 6098-6101, December 1978 Ten-nanometer filaments of hamster BHK-21 cells and epidermal filaments have similar structures (electron microscopy/spectropolarimetry/wide-angle x-ray diffraction) PETER M. STEINERT"t, STEVEN B. ZIMMERMAN*, JUDITH M. STARGER§, AND ROBERT D. GOLDMAN§ *Dermatology Branch, National Cancer Institute; *Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; and §Department of Biological Sciences, Carnegie-Mellon University, 4400 Fifth-Avenue, Pittsburgh, Pennsylvania 15213 Communicated by David R. Davies, September 22,1978

ABSTRACT The 10-nm filaments of baby hamster kidney structure of keratin filaments from both (and ) and (BHK-21) cells, when examined either in the form of native is now well advanced (26, 27). Thus consideration ilanent caps or polymerized in vitro, are long tubes of 8-10 nm in diameter. They contain about 42% a-helix, which, and comparison of the properties-of keratin filaments with those on the basis of x-ray diffraction data, is arranged n a coiled-coil of the similar-sized filaments from other cell types may be conformation characteristic of of the a type. The known useful. Keratin filaments are a-helix-rich fibrous proteins that structural properties such as morphology, dimensions, subunit exhibit an a type x-ray diffraction pattern characteristic of composition, and ultrastructure of this fibrous protein are very proteins of the k ("hard" keratin)-m ()-e (epidermin similar to those of the mammalian epidermal keratin filament, = epidermal keratin)-f (fibrin) class (19, 28). This x-ray dif- to which it may therefore be related. fraction pattern has been interpreted in terms of models in It is now apparent that all eukaryotic cell types contain at least which a-helical regions of the subunits are arranged in a two- three different types of intracellular structural proteins: mi- or three-strandedl supercoiled or coiled-coil conformation (29, crotubules (about 25 nm in diameter), -containing mi- 30). Direct evidence for this concept stems from the more recent crofilaments (about 5-7 nm in diameter), and a less-charac- characterization of a-helix-rich regions containing two (31, 32) terized class of filaments of intermediate dimensions (7-12 nm or three coiled-coil chains (27, 33, 34) in the repeating structural in diameter). The latter have been referred to as intermediate units of several a-type proteins. Interestingly, one report has or 10-nm filaments (1-7) or specifically as in demonstrated that whole axoplasm of Myxicola, of which neuronal tissues (8-13) and have been termed skeletin (14) or neurofilaments are the major component, gives an a type x-ray (15) in smooth muscle. Filaments of similar dimensions diffraction pattern (35). This finding suggests that a comparison keratin- of the 10-nm filaments of different cell types with keratin are prevalent in epithelial tissues (16-18), including filaments may indeed be of interest. producing cells of epidermis and such epidermal Recently, procedures have been developed for the rapid derivatives as hair or wool (19). These filaments are more isolation of 10-nm filaments from baby hamster kidney commonly referred to as tonofilaments or keratin filaments. (BHK-21) cells grown in culture in amounts sufficient for de- In most cell types, the intermediate filaments are thought to tailed chemical and structural studies (17, 18). In this report, provide a relatively stable cytoskeletal framework within the are of the a and cells and to be involved in such functions as maintenance of cell we demonstrate that these filaments type shape (20), intracellular transport (3. 9), organelle attachment suggest that they are structurally similar to epidermal keratin or movement (6, 14, 15, 21,22), and cell locomotion in cultured filaments. cells (2, 17, 18). In more specialized cells such as , MATERIALS AND METHODS these filaments comprise up to 70% of the total cellular mass (19, 23) and, through interconnections between desmosomes, Isolation of 10-nm Filaments. Spreading populations of appear to lend a rigid or flexible texture to the tissue (19, BHK-21 cells contain juxtanuclear caps of 10-nm filaments 24). (FC). These were harvested from the cells as described previ- The grouping of 10-nm filaments, neurofilaments, tonofi- ously (17, 18), and the pellet of FC was washed in buffer con- laments and other intermediate filaments into a class of similar taining 6 mM Na+/K+ phosphate (pH 7.4), 171 mM NaCl, 3 fibrous proteins has been based almost exclusively on similarities mM KCl, and 0.5 mM phenylmethylsulfonyl fluoride (Sigma). in morphology and compositions of major subunits The FC were resuspended in a small volume (protein concen- (4, 17). Therefore, it is of considerable importance to use more tration about 1 mg/ml) of 6 mM Na+/K+ phosphate (pH 7.4) quantitative chemical and structural techniques to determine containing 0.1 mM phenylmethylsulfor~yl fluoride and dialyzed the extent of homology within this class from different cell against 1000 vol of this buffer at 40O fr 18 hr to disperse the types. In the cells studied to date, the reported numbers of filaments into protofilamentous unit (i7). The opaque solution filament subunits vary from one to a large number, and their was centrifuged at 40,000 X g for 30 min and then at 250,000 molecular weights are within the range 45,000-212,000 (4,5). X g for 1 hr. The resulting clear supernatant contained more However, it is unclear whether these differences are real, as than 75% of the total FC protein. On addition of 3 M NaCl to suggested by peptide mapping (25), or artifacts due to peptide a final concentration of 0.17 M filaments polymerized in vitro degradation (13). within 6 hr at 40C. The filaments were obtained as a pellet after While comparatively little is known of the structure of this centrifugation at 100,000 X g for 45 min. When required, the class of filaments from most cell types, knowledge of the pellets were resuspended by gentle homogenization in 6 mM Na+/K+ phosphate buffer (pH 7.4) containing 0.1 mM phen- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: FC, filament caps; ORD, optical rotatory dispersion; vertisement" in accordance with 18 U. S. C. §1734 solely to indicate CD, circular dichroism. this fact. t To whom all correspondence should be addressed. 6098 Downloaded by guest on September 29, 2021 Cell Biology: Steinert et al. Proc. Natl. Acad. Sc. USA 75 (1978) 6099


A i I P. v

4L A


FIG. 1. Structure of repolymerized filaments. Filaments were negatively stained with (A) uranyl acetate (X95,000) or (B) neutralized phosphotungstic acid (X240,000). (C) Transverse cross-section through a fiber used for x-ray diffraction. (X240,000.) In A-C, the bar is (D) Nine percent T/3% C sodium dodecyl sulfate-polyacrylamide gel. ylmethylsulfonyl fluoride, dialyzed against 1000 vol of this crocamera with a specimen-to-film distance of 14-36 mm and buffer for 18 hr at 40C, clarified by centrifugation, and re- exposed to Cu-Ka radiation (X = 1.54 A) in an atmosphere of polymerized on addition of 3 M NaCl to 0.17 M. dry helium for up to 48 hr. The 2.82-A and 3.27-A diffraction Analytical Procedures. Protein was estimated by the method rings from the NaCl present permitted calibration of the dif- of Bramhall et al. (36). Polyacrylamide gel electrophoresis was fraction patterns. performed by using a multiphasic system with 0:1% sodium dodecyl sulfate on 9% T/3% C gels (37). RESULTS Electron Microscopy. Specimens were diluted to a protein BHK-21 filaments can be polymerized in vitro by addition of concentration of about 20 Ag/ml with 6 mM Na+/K+ phos- NaCl to a final concentration of 0.17 M to a solution of solubi- phate buffer (pH 7.4) and examined on carbon-coated lized 10-nm filament caps (17). On pelleting from suspension, ("stress-free") grids (Ladd, Burlington, VT) after negative these filaments can be disassembled with low ionic strength with either 0.7% uranyl acetate or 1% phosphotungstic phosphate buffer and repolymerized on addition of NaCl to acid neutralized to pH 6.8 with KOH (38). Fibers used for x-ray 0.17 M, with a yield of at least 50% of the starting protein. On diffraction were fixed in phosphate-Ixiffered glutaraldehyde, negative staining, these filaments range in diameter from 8 to postfixed in OS04, and embedded, and ultrathin sections were 10 nm, are about 1 gm long, and appear to have a dense-staining stained on the grid with uranyl acetate and lead citrate (17, core throughout their length (Fig. 1 A and B). In transverse 18). cross-section, they possess a region of diminished electron Estimation of a-Helix Contents. Both optical rotatory dis- density in their centers suggestive of a tubular structure (Fig. persion (ORD) and circular dichroism (CD) were used. Samples iC); this appearance is similar to their appearance in BHK-21 for measurement were equilibrated at 0.1-0.2 mg/nil in 6mM cells in 3itu (43). On dissociation with sodium dodecyl sulfate Na+/K+ phosphate buffer (pH 7.4). Measurements were made followed by polyacrylamide gel electrophoresis, more than 95% at 230C in 1-cm quartz cells with a Cary model 60 spectropo- of the protein associated with the repolymerized filaments larimeter equipped with a model 6001 CD accessory. In ORD appears as two bands of molecular weights about 55,000 and studies, the a-helix content was estimated from the mean res- 54,000 (Fig. ID). Minor bands of molecular weights about idue rotation observed at 233 nm by using values of -1800° for 200,000 and 52,000 are also present. 0% a-helix and -12,000° for 100% a-helix, with presumption By ORD, solubilized FC or repolymerized filaments have of linear interpolation (39). Values for bo were also calculated a bo value of -265 and a mean residue rotation at 233 nm of from Moffit plots of Xo = 212 nm and were reduced to a-helix -5900°. These values correspond to an a-helix content of contents by using the formula (bo/-630) X 100% (40). In CD 40-42%. By CD, their mean molar ellipticity value of studies, the mean molar ellipticity values at 208 nm were used -12,400°cn2.dmol-I at 208 nm corresponds to an a-helix to calculate the a-helix contents from values of 4000° cm2- content of 44%. dmolh' for and -33,000° cm2.dmohl- for 100% Wide-angle x-ray diffraction analyses of fibers prepared from a-helix (41). pellets of repolymerized filaments show the presence of X-Ray Diffraction. A pellet of repolymerized filaments was equatorial spots at 9.7 A and meridional arcs at 5.17 A (Fig. 2). used to draw fibers of 20-30 Am diameter (42). Fibers were The latter reflections are significantly less than the 5.4-A chosen whose birefringence suggested a high degree of align- spacing expected of a normal a-helix and thereby characterize ment of the filaments. A fiber was mounted in a Norelco mi- this pattern as of the a type. Downloaded by guest on September 29, 2021 6100 Cell Biology: Steinert et al. Proc. Natl. Acad. Sci. USA 75 (1978) An earlier report demonstrated that Myxicola neurofilaments are also of the a type (35). This finding, together with the present results and the observation that the major subunits of intermediate-sized filaments of a variety of cell types have similar amino acid compositions (4, 17), suggests that there are common structural and chemical features within this size class of cytoplasmic filaments. Such similarities serve as a more rigorous distinction between intermediate filaments and the smaller and larger , neither of which is of the a type. Moreover, these conclusions should provide a focus for additional comparative and structural studies of intermediate filaments of other cell types. We thank Dr. Marisa Gullino for expert assistance with the electron microscopy. This work was supported in part by a grant (to R.D.G.) from the National Science Foundation. 1. Goldman, R. D., Pollard, T. D. & Rosenbaum, J. L., eds. (1976) Cell Motility, Cold Spring Harbor Conferences on Cell Prolif- eration (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 3. 2. Goldman, R. D., Berg, G., Bushnell, A., Chang, C. M., Dickerman, L. H., Hopkins, N., Miller, M. L., Pollack, R. & Wang, E. (1973) in Ciba Foundation Symposium, No. 14, (Elsevier/North-Hol- land, New York), pp. 83-107. 3. Goldman, R. D. & Knipe, D. M. (1973) Cold Spring Harbor Symp. Quant. Biol. 37,523-534. FIG. 2. Wide-angle x-ray diffraction pattern. Equatorial spots 4. Goldman, R. D., Milsted, A., Schloss, J. A., Starger, J. M. & Yerna, and meridional arcs are evident at 9.7 A and 5.17 A, respectively. The M.-J. (1979) Annu. Rev. Physiol., in press. diffraction ring (at the corners) represents the 3.27-A spacing of 5. Gilbert, D. S. (1978) Nature (London) 272,577-578. NaCl. 6. Cooke, P. H. (1976) J. Cell Biol. 68,539-556. 7. Hynes, R. 0. & Destree, A. T. (1978) Cell 13, 151-163. 8. Huneeus, F. & Davison, P. (1970) J. Mol. Biol. 52,415-428. DISCUSSION 9. Lasek, R. J. & Hoffman, P. N. (1976) in Cell Motility, Cold Spring Harbor Conferences on Cell Proliferation, eds. Goldman, R. D., The present experiments demonstrate that the 10-nm filaments Pollard, T. D. & Rosenbaum, J. L. (Cold Spring Harbor Labo- of BHK-21 cells, when seen in situ or after disassembly-reas- ratory, Cold Spring Harbor, NY), Vol. 3, Book C, pp. 1021- sembly in vitro, are long tubular structures 8-10 nm in diameter 1050. and contain 40-44% a-helix. On the basis of the x-ray diffrac- 10. Schlaepfer, W. (1977) J. Cell Biol. 74,226-240. tion pattern given by the filaments, this a-helix is arranged in 11. Yen, S. H., Dahl, D., Schachner, M. & Shelanski, M. L. (1976) a coiled-coil conformation characteristic of a type proteins of Proc. Natl. Acad. Sci. USA 73,529-533. the k-m-e-f class. In each a type protein so far studied, this 12. Shelanski, M. L., Yen, S. H. & Lee, V. M. (1976) in Cell Motility, type of structure is formed by the coiling of a-helices on either Cold Spring Harbor Conferences on Cell Proliferation, eds. two or three adjacent subunits in the repeating structural unit Goldman, R. D., Pollard, T. D. & Rosenbaum, J. L. (Cold Spring of the fibrous protein. However, it remains to be determined Harbor Laboratory, Cold Spring Harbor, NY), Vol. 3, Book C, in BHK-21 10-nm filaments whether the coiled-coil consists of pp. 1007-1020. two or three chains and whether the coiled-coil exists as a con- 13. Gilbert, D. S., Newby, B. J. & Anderton, B. H. (1975) Nature (London) 256,586-589. tinuous region as in myosin (31), or is segmented (interspersed 14. Small, J. V. & Sobieszek, A. (1977) J. Cell Sci. 23, 243-268. by regions of as in non-a-helix) epidermal (27) and wool (33) 15. Izant, J. & Lazarides, E. (1977) Proc. Natl. Acad. Sci. USA 74, keratin filaments. 1450-1454. The BHK-21 10-nm filaments share several properties with 16. Brecher, S. (1975) Exp. Cell Res. 96,303-310. mammalian epidermal keratin filaments (42): the two have 17. Starger, J. M., Brown, W. E., Goldman, A. E. & Goldman, R. D. similar dimensions both in vivo and when polymerized in vitro; (1978) J. Cell Biol. 78,93-109. both appear as tubes when negatively stained or in cross-section 18. Starger, J. M. & Goldman, R. D. (1976) Proc. Natl. Acad. Sci. USA (P. M. Steinert, unpublished observations); both possess an a 74,2422-2426. type ultrastructure; and their subunit compositions are similar 19. Fraser, R. B. D., MacRae, T. M. & Rogers, G. E. (1972) , in terms of net charge, molecular weight, and amino acid Their Composition, Structure and Biosynthesis (Thomas, content, although the subunits of epidermal keratin filaments Springfield, IL). appear to be more heterogeneous. In addition, BHK-21 10-nm 20. Goldman, R. D. & Follet, E. A. C. (1970) Science 169, 286- filaments are likewise similar to the keratin filaments of hair 288. and wool (19) and the filaments of the cells 21. Goldman, R. D. (1971) J. Cell Biol. 51,752-762. 22. Lehto, V.-P., Virtanen, I. & Kurki, P. (1978) Nature (London) of the hair follicle (44, 45), except for certain distinctive dif- 272, 175-177. ferences in amino acid contents. Such apparent similarities to 23. Steinert, P. M. (1975) Biochem. J. 149,39-48. the filaments of these keratinizing tissues suggest that further 24. Matoltsy, A. G. (1975) J. Invest. Dermatol. 65, 127-142. structural features and properties of the 10-nm filaments may 25. Davison, P., Hong, B. S. & Cooke, P. H. (1977) Exp. Cell Res. 109, be common. The only notable difference between BHK-21 471-474. filaments and keratin filaments is their properties. 26. Fraser, R. D. B., MacRae, T. P. & Suzuki, E. (1976) J. Mol. Biol. Whereas epidermal keratin filaments may only be polymerized 108,435-452. in vitro in low ionic strength salt solutions (42), the BHK-21 27. Steinert, P. M. (1978) J. Mol. Biol. 123,49-70. 10-nm filaments dissociate under these conditions and poly- 28. Bailey, C. J., Astbury, W. T. & Rudall, K. M. (1943) Nature merize near physiological ionic conditions. (London) 151, 716-717. Downloaded by guest on September 29, 2021 Cell Biology: Steinert et al. Proc. Nati. Acad. Sci. USA 75 (1978) 6101

29. Pauling, L. & Corey, R. B. (1953) Nature (London) 171, 59- 36. Bramhall, S., Noack, N., Wu, M. & Lowenberg, J. R. (1969) Anal. 61. ~Bichem. 31,146-148. 30. Crick, F. H. C. (1953) Acta Crystallogr. 6,685-688. 37. Steinert, P. M. & Idler, W. W. (1975) Biochem. J. 151, 603- 31. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969) J. Mol. 614. Biol. 42, 1-29. 38. Jones, L. N. (1976) Biochim. Biophys. Acta 446,515-524. 32. Hodges, R. S., Sodek, J., Smillie, S. B. & Jurasek, L. (1972) Cold 39. Simons, N. S., Cohen, C., Szent-Gyorgyi, A. G., Wetlaufer, D. B. Spring Harbor Symp. Quant. Biol. 32,299-310. & Blout, E. R. (1961) J. Am. Chem. Soc. 83,4766-4769. 40. Blout, E. R. (1960) in Optical Rotatory Dispersion, ed. Djerassi, 33. Crewther, W. G., Dowling, L. M., Gough, K. H., Inglis, A. S., McKern, N. M., Sparrow, L. G. & Woods, E. F. (1976) in Pro- C. (McGraw-Hill, New York), pp. 248-279. Research 41. Greenfield, N. & Fasman, G. D. (I 969) Biochemistry 8, 4108- ceedings of the 5th International Wool and Textiles 4116. Conference, Aachen, Germany, ed. Ziegler, K. (Dtsch. Woll- 42. Steinert, P. M., Idler, W. W. & Zitinerman, S. B. (1976) J. Mol. forschungsinstitut Tech. Hochsch., Aachen, Germany), Vol. 2, Biol. 108, 547-567. pp. 233-242. 43. Goldman, R. D. & Follet, E. A. C. (1969) Exp. Cell Res. 57, 34. Doolittle, R. F., Goldbaum, D. S. & Doolittle, L. R. (1978) J. Mol. 263-276. Biol. 120,311-325. 44. Steinert, P. M., Dyer, P. Y.. & Rogers, G. E. (1971) J. Invest. 35. Day, W. A. & Gilbert, D. S. (1972) Biochim. Biophys. Acta 285, Dermatol. 56, 49-54. 503-506. 45. Steinert, P. M. (1978) Biochemistry 17, in press. Downloaded by guest on September 29, 2021