Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 6098-6101, December 1978
Cell Biology
Ten-nanometer filaments of hamster BHK-21 cells and epidermal keratin 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 (BHK-21) cells, when examined either in the form of native ilanent caps or polymerized in vitro, are long tubes of protein 8-10 nm in diameter. They contain about 42% a-helix, which, on the basis of x-ray diffraction data, is arranged n a coiled-coil conformation characteristic of proteins of the a type. The known structural properties such as morphology, dimensions, subunit composition, and ultrastructure of this fibrous protein are very similar to those of the mammalian epidermal keratin filament, to which it may therefore be related. It is now apparent that all eukaryotic cell types contain at least three different types of intracellular structural proteins: microtubules (about 25 nm in diameter), actin-containing microfilaments (about 5-7 nm in diameter), and a less-characterized class of filaments of intermediate dimensions (7-12 nm in diameter). The latter have been referred to as intermediate or 10-nm filaments (1-7) or specifically as neurofilaments in neuronal tissues (8-13) and have been termed skeletin (14) or desmin (15) in smooth muscle. Filaments of similar dimensions are prevalent in epithelial tissues (16-18), including keratinproducing cells of vertebrate epidermis and such epidermal derivatives as hair or wool (19). These filaments are more commonly referred to as tonofilaments or keratin filaments. In most cell types, the intermediate filaments are thought to provide a relatively stable cytoskeletal framework within the cells and to be involved in such functions as maintenance of cell shape (20), intracellular transport (3. 9), organelle attachment or movement (6, 14, 15, 21,22), and cell locomotion in cultured cells (2, 17, 18). In more specialized cells such as keratinocytes, these filaments comprise up to 70% of the total cellular mass (19, 23) and, through interconnections between desmosomes, appear to lend a rigid or flexible texture to the tissue (19, 24). The grouping of 10-nm filaments, neurofilaments, tonofilaments and other intermediate filaments into a class of similar fibrous proteins has been based almost exclusively on similarities in morphology and amino acid compositions of major subunits (4, 17). Therefore, it is of considerable importance to use more quantitative chemical and structural techniques to determine the extent of homology within this class from different cell types. In the cells studied to date, the reported numbers of filament subunits vary from one to a large number, and their molecular weights are within the range 45,000-212,000 (4,5 ). However, it is unclear whether these differences are real, as suggested by peptide mapping (25), or artifacts due to peptide
degradation (13). While comparatively little is known of the structure of this class of filaments from most cell types, knowledge of the
structure of keratin filaments from both hair (and wool) and epidermis is now well advanced (26, 27). Thus consideration and comparison of the properties-of keratin filaments with those of the similar-sized filaments from other cell types may be useful. Keratin filaments are a-helix-rich fibrous proteins that exhibit an a type x-ray diffraction pattern characteristic of proteins of the k ("hard" keratin)-m (myosin)-e (epidermin = epidermal keratin)-f (fibrin) class (19, 28). This x-ray diffraction pattern has been interpreted in terms of models in which a-helical regions of the subunits are arranged in a twoor three-strandedl supercoiled or coiled-coil conformation (29, 30). Direct evidence for this concept stems from the more recent characterization of a-helix-rich regions containing two (31, 32) or three coiled-coil chains (27, 33, 34) in the repeating structural units of several a-type proteins. Interestingly, one report has demonstrated that whole axoplasm of Myxicola, of which neurofilaments are the major component, gives an a type x-ray diffraction pattern (35). This finding suggests that a comparison of the 10-nm filaments of different cell types with keratin filaments may indeed be of interest. Recently, procedures have been developed for the rapid isolation of 10-nm filaments from baby hamster kidney (BHK-21) cells grown in culture in amounts sufficient for detailed chemical and structural studies (17, 18). In this report, we demonstrate that these filaments are of the a type and suggest that they are structurally similar to epidermal keratin filaments. MATERIALS AND METHODS Isolation of 10-nm Filaments. Spreading populations of BHK-21 cells contain juxtanuclear caps of 10-nm filaments (FC). These were harvested from the cells as described previously (17, 18), and the pellet of FC was washed in buffer containing 6 mM Na+/K+ phosphate (pH 7.4), 171 mM NaCl, 3 mM KCl, and 0.5 mM phenylmethylsulfonyl fluoride (Sigma). The FC were resuspended in a small volume (protein concentration about 1 mg/ml) of 6 mM Na+/K+ phosphate (pH 7.4) containing 0.1 mM phenylmethylsulfor~yl fluoride and dialyzed against 1000 vol of this buffer at 40O fr 18 hr to disperse the filaments into protofilamentous unit (i7). The opaque solution was centrifuged at 40,000 X g for 30 min and then at 250,000 X g for 1 hr. The resulting clear supernatant contained more than 75% of the total FC protein. On addition of 3 M NaCl to a final concentration of 0.17 M filaments polymerized in vitro within 6 hr at 40C. The filaments were obtained as a pellet after centrifugation at 100,000 X g for 45 min. When required, 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 "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: FC, filament caps; ORD, optical rotatory dispersion; CD, circular dichroism. t To whom all correspondence should be addressed. 6098
Cell Biology: Steinert et al.
Proc. Natl. Acad. Sc. USA 75 (1978)
I
6099
Jr Ai P. v
4L
A
I
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 0. 1.um. (D) Nine percent T/3% C sodium dodecyl sulfate-polyacrylamide gel.
ylmethylsulfonyl fluoride, dialyzed against 1000 vol of this buffer for 18 hr at 40C, clarified by centrifugation, and repolymerized on addition of 3 M NaCl to 0.17 M. Analytical Procedures. Protein was estimated by the method of Bramhall et al. (36). Polyacrylamide gel electrophoresis was performed by using a multiphasic system with 0:1% sodium dodecyl sulfate on 9% T/3% C gels (37). Electron Microscopy. Specimens were diluted to a protein concentration of about 20 Ag/ml with 6 mM Na+/K+ phosphate buffer (pH 7.4) and examined on carbon-coated ("stress-free") grids (Ladd, Burlington, VT) after negative staining with either 0.7% uranyl acetate or 1% phosphotungstic acid neutralized to pH 6.8 with KOH (38). Fibers used for x-ray diffraction were fixed in phosphate-Ixiffered glutaraldehyde, postfixed in OS04, and embedded, and ultrathin sections were stained on the grid with uranyl acetate and lead citrate (17, 18). Estimation of a-Helix Contents. Both optical rotatory dispersion (ORD) and circular dichroism (CD) were used. Samples for measurement were equilibrated at 0.1-0.2 mg/nil in 6 mM Na+/K+ phosphate buffer (pH 7.4). Measurements were made at 230C in 1-cm quartz cells with a Cary model 60 spectropolarimeter equipped with a model 6001 CD accessory. In ORD studies, the a-helix content was estimated from the mean residue rotation observed at 233 nm by using values of -1800° for 0% a-helix and -12,000° for 100% a-helix, with presumption of linear interpolation (39). Values for bo were also calculated from Moffit plots of Xo = 212 nm and were reduced to a-helix contents by using the formula (bo/-630) X 100% (40). In CD studies, the mean molar ellipticity values at 208 nm were used to calculate the a-helix contents from values of 4000° cm2dmolh' for random coil and -33,000° cm2.dmohl- for 100% a-helix (41). X-Ray Diffraction. A pellet of repolymerized filaments was used to draw fibers of 20-30 Am diameter (42). Fibers were chosen whose birefringence suggested a high degree of alignment of the filaments. A fiber was mounted in a Norelco mi-
crocamera with a specimen-to-film distance of 14-36 mm and exposed to Cu-Ka radiation (X = 1.54 A) in an atmosphere of dry helium for up to 48 hr. The 2.82-A and 3.27-A diffraction rings from the NaCl present permitted calibration of the diffraction patterns. RESULTS BHK-21 filaments can be polymerized in vitro by addition of NaCl to a final concentration of 0.17 M to a solution of solubilized 10-nm filament caps (17). On pelleting from suspension, these filaments can be disassembled with low ionic strength phosphate buffer and repolymerized on addition of NaCl to 0.17 M, with a yield of at least 50% of the starting protein. On negative staining, these filaments range in diameter from 8 to 10 nm, are about 1 gm long, and appear to have a dense-staining core throughout their length (Fig. 1 A and B). In transverse cross-section, they possess a region of diminished electron density in their centers suggestive of a tubular structure (Fig. iC); this appearance is similar to their appearance in BHK-21 cells in 3itu (43). On dissociation with sodium dodecyl sulfate followed by polyacrylamide gel electrophoresis, more than 95% of the protein associated with the repolymerized filaments appears as two bands of molecular weights about 55,000 and 54,000 (Fig. ID). Minor bands of molecular weights about 200,000 and 52,000 are also present. By ORD, solubilized FC or repolymerized filaments have a bo value of -265 and a mean residue rotation at 233 nm of -5900°. These values correspond to an a-helix content of 40-42%. By CD, their mean molar ellipticity value of -12,400°cn2.dmol-I at 208 nm corresponds to an a-helix content of 44%. Wide-angle x-ray diffraction analyses of fibers prepared from pellets of repolymerized filaments show the presence of equatorial spots at 9.7 A and meridional arcs at 5.17 A (Fig. 2). The latter reflections are significantly less than the 5.4-A spacing expected of a normal a-helix and thereby characterize this pattern as of the a type.
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 microfilaments and larger microtubules, 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.
FIG. 2.
Wide-angle
x-ray
diffraction pattern. Equatorial
spots
and meridional arcs are evident at 9.7 A and 5.17 A, respectively. The diffraction ring (at the corners) represents the 3.27-A spacing of NaCl.
DISCUSSION The present experiments demonstrate that the 10-nm filaments of BHK-21 cells, when seen in situ or after disassembly-reassembly in vitro, are long tubular structures 8-10 nm in diameter and contain 40-44% a-helix. On the basis of the x-ray diffraction pattern given by the filaments, this a-helix is arranged in a coiled-coil conformation characteristic of type proteins of the k-m-e-f class. In each a type protein so far studied, this type of structure is formed by the coiling of a-helices on either two or three adjacent subunits in the repeating structural unit of the fibrous protein. However, it remains to be determined in BHK-21 10-nm filaments whether the coiled-coil consists of two or three chains and whether the coiled-coil exists as a continuous region as in myosin (31), or is segmented (interspersed by regions of non-a-helix) as in epidermal (27) and wool (33) keratin filaments. The BHK-21 10-nm filaments share several properties with mammalian epidermal keratin filaments (42): the two have similar dimensions both in vivo and when polymerized in vitro; both appear as tubes when negatively stained or in cross-section a
(P. M. Steinert, unpublished observations); both
possess an
a
type ultrastructure; and their subunit compositions are similar in terms of net charge, molecular weight, and amino acid content, although the subunits of epidermal keratin filaments appear to be more heterogeneous. In addition, BHK-21 10-nm filaments are likewise similar to the keratin filaments of hair and wool (19) and the filaments of the inner root sheath cells of the hair follicle (44, 45), except for certain distinctive dif-
ferences in amino acid contents. Such apparent similarities to the filaments of these keratinizing tissues suggest that further structural features and properties of the 10-nm filaments may be common. The only notable difference between BHK-21 filaments and keratin filaments is their solubility properties. Whereas epidermal keratin filaments may only be polymerized low ionic strength salt solutions (42), the BHK-21 10-nm filaments dissociate under these conditions and polymerize near physiological ionic conditions.
in vitro in
1. Goldman, R. D., Pollard, T. D. & Rosenbaum, J. L., eds. (1976) Cell Motility, Cold Spring Harbor Conferences on Cell Proliferation (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-Holland, New York), pp. 83-107. 3. Goldman, R. D. & Knipe, D. M. (1973) Cold Spring Harbor Symp. Quant. Biol. 37,523-534. 4. Goldman, R. D., Milsted, A., Schloss, J. A., Starger, J. M. & Yerna, M.-J. (1979) Annu. Rev. Physiol., in press. 5. Gilbert, D. S. (1978) Nature (London) 272,577-578. 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. 9. Lasek, R. J. & Hoffman, P. N. (1976) in Cell Motility, Cold Spring Harbor Conferences on Cell Proliferation, eds. Goldman, R. D., Pollard, T. D. & Rosenbaum, J. L. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 3, Book C, pp. 10211050. 10. Schlaepfer, W. (1977) J. Cell Biol. 74,226-240. 11. Yen, S. H., Dahl, D., Schachner, M. & Shelanski, M. L. (1976) Proc. Natl. Acad. Sci. USA 73,529-533. 12. Shelanski, M. L., Yen, S. H. & Lee, V. M. (1976) in Cell Motility, Cold Spring Harbor Conferences on Cell Proliferation, eds. Goldman, R. D., Pollard, T. D. & Rosenbaum, J. L. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 3, Book C, pp. 1007-1020. 13. Gilbert, D. S., Newby, B. J. & Anderton, B. H. (1975) Nature (London) 256,586-589. 14. Small, J. V. & Sobieszek, A. (1977) J. Cell Sci. 23, 243-268. 15. Izant, J. & Lazarides, E. (1977) Proc. Natl. Acad. Sci. USA 74, 1450-1454. 16. Brecher, S. (1975) Exp. Cell Res. 96,303-310. 17. Starger, J. M., Brown, W. E., Goldman, A. E. & Goldman, R. D. (1978) J. Cell Biol. 78,93-109. 18. Starger, J. M. & Goldman, R. D. (1976) Proc. Natl. Acad. Sci. USA 74,2422-2426. 19. Fraser, R. B. D., MacRae, T. M. & Rogers, G. E. (1972) Keratins, Their Composition, Structure and Biosynthesis (Thomas, Springfield, IL). 20. Goldman, R. D. & Follet, E. A. C. (1970) Science 169, 286288. 21. Goldman, R. D. (1971) J. Cell Biol. 51,752-762. 22. Lehto, V.-P., Virtanen, I. & Kurki, P. (1978) Nature (London) 272, 175-177. 23. Steinert, P. M. (1975) Biochem. J. 149,39-48. 24. Matoltsy, A. G. (1975) J. Invest. Dermatol. 65, 127-142. 25. Davison, P., Hong, B. S. & Cooke, P. H. (1977) Exp. Cell Res. 109, 471-474. 26. Fraser, R. D. B., MacRae, T. P. & Suzuki, E. (1976) J. Mol. Biol. 108,435-452. 27. Steinert, P. M. (1978) J. Mol. Biol. 123,49-70. 28. Bailey, C. J., Astbury, W. T. & Rudall, K. M. (1943) Nature (London) 151, 716-717.
Cell Biology: Steinert et al. 29. Pauling, L. & Corey, R. B. (1953) Nature (London) 171, 5961. 30. Crick, F. H. C. (1953) Acta Crystallogr. 6,685-688. 31. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969) J. Mol. Biol. 42, 1-29. 32. Hodges, R. S., Sodek, J., Smillie, S. B. & Jurasek, L. (1972) Cold Spring Harbor Symp. Quant. Biol. 32,299-310. 33. Crewther, W. G., Dowling, L. M., Gough, K. H., Inglis, A. S., McKern, N. M., Sparrow, L. G. & Woods, E. F. (1976) in Proceedings of the 5th International Wool and Textiles Research Conference, Aachen, Germany, ed. Ziegler, K. (Dtsch. Wollforschungsinstitut Tech. Hochsch., Aachen, Germany), Vol. 2, pp. 233-242. 34. Doolittle, R. F., Goldbaum, D. S. & Doolittle, L. R. (1978) J. Mol. Biol. 120,311-325. 35. Day, W. A. & Gilbert, D. S. (1972) Biochim. Biophys. Acta 285, 503-506.
Proc. Nati. Acad. Sci. USA 75 (1978)
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36. Bramhall, S., Noack, N., Wu, M. & Lowenberg, J. R. (1969) Anal.
~Bichem. 31,146-148. 37. Steinert, P. M. & Idler, W. W. (1975) Biochem. J. 151, 603614. 38. Jones, L. N. (1976) Biochim. Biophys. Acta 446,515-524. 39. Simons, N. S., Cohen, C., Szent-Gyorgyi, A. G., Wetlaufer, D. B. & Blout, E. R. (1961) J. Am. Chem. Soc. 83,4766-4769. 40. Blout, E. R. (1960) in Optical Rotatory Dispersion, ed. Djerassi, C. (McGraw-Hill, New York), pp. 248-279. 41. Greenfield, N. & Fasman, G. D. (I 969) Biochemistry 8, 41084116. 42. Steinert, P. M., Idler, W. W. & Zitinerman, S. B. (1976) J. Mol. Biol. 108, 547-567. 43. Goldman, R. D. & Follet, E. A. C. (1969) Exp. Cell Res. 57, 263-276. 44. Steinert, P. M., Dyer, P. Y.. & Rogers, G. E. (1971) J. Invest. Dermatol. 56, 49-54. 45. Steinert, P. M. (1978) Biochemistry 17, in press.
Cell Biology
Ten-nanometer filaments of hamster BHK-21 cells and epidermal keratin 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 (BHK-21) cells, when examined either in the form of native ilanent caps or polymerized in vitro, are long tubes of protein 8-10 nm in diameter. They contain about 42% a-helix, which, on the basis of x-ray diffraction data, is arranged n a coiled-coil conformation characteristic of proteins of the a type. The known structural properties such as morphology, dimensions, subunit composition, and ultrastructure of this fibrous protein are very similar to those of the mammalian epidermal keratin filament, to which it may therefore be related. It is now apparent that all eukaryotic cell types contain at least three different types of intracellular structural proteins: microtubules (about 25 nm in diameter), actin-containing microfilaments (about 5-7 nm in diameter), and a less-characterized class of filaments of intermediate dimensions (7-12 nm in diameter). The latter have been referred to as intermediate or 10-nm filaments (1-7) or specifically as neurofilaments in neuronal tissues (8-13) and have been termed skeletin (14) or desmin (15) in smooth muscle. Filaments of similar dimensions are prevalent in epithelial tissues (16-18), including keratinproducing cells of vertebrate epidermis and such epidermal derivatives as hair or wool (19). These filaments are more commonly referred to as tonofilaments or keratin filaments. In most cell types, the intermediate filaments are thought to provide a relatively stable cytoskeletal framework within the cells and to be involved in such functions as maintenance of cell shape (20), intracellular transport (3. 9), organelle attachment or movement (6, 14, 15, 21,22), and cell locomotion in cultured cells (2, 17, 18). In more specialized cells such as keratinocytes, these filaments comprise up to 70% of the total cellular mass (19, 23) and, through interconnections between desmosomes, appear to lend a rigid or flexible texture to the tissue (19, 24). The grouping of 10-nm filaments, neurofilaments, tonofilaments and other intermediate filaments into a class of similar fibrous proteins has been based almost exclusively on similarities in morphology and amino acid compositions of major subunits (4, 17). Therefore, it is of considerable importance to use more quantitative chemical and structural techniques to determine the extent of homology within this class from different cell types. In the cells studied to date, the reported numbers of filament subunits vary from one to a large number, and their molecular weights are within the range 45,000-212,000 (4,5 ). However, it is unclear whether these differences are real, as suggested by peptide mapping (25), or artifacts due to peptide
degradation (13). While comparatively little is known of the structure of this class of filaments from most cell types, knowledge of the
structure of keratin filaments from both hair (and wool) and epidermis is now well advanced (26, 27). Thus consideration and comparison of the properties-of keratin filaments with those of the similar-sized filaments from other cell types may be useful. Keratin filaments are a-helix-rich fibrous proteins that exhibit an a type x-ray diffraction pattern characteristic of proteins of the k ("hard" keratin)-m (myosin)-e (epidermin = epidermal keratin)-f (fibrin) class (19, 28). This x-ray diffraction pattern has been interpreted in terms of models in which a-helical regions of the subunits are arranged in a twoor three-strandedl supercoiled or coiled-coil conformation (29, 30). Direct evidence for this concept stems from the more recent characterization of a-helix-rich regions containing two (31, 32) or three coiled-coil chains (27, 33, 34) in the repeating structural units of several a-type proteins. Interestingly, one report has demonstrated that whole axoplasm of Myxicola, of which neurofilaments are the major component, gives an a type x-ray diffraction pattern (35). This finding suggests that a comparison of the 10-nm filaments of different cell types with keratin filaments may indeed be of interest. Recently, procedures have been developed for the rapid isolation of 10-nm filaments from baby hamster kidney (BHK-21) cells grown in culture in amounts sufficient for detailed chemical and structural studies (17, 18). In this report, we demonstrate that these filaments are of the a type and suggest that they are structurally similar to epidermal keratin filaments. MATERIALS AND METHODS Isolation of 10-nm Filaments. Spreading populations of BHK-21 cells contain juxtanuclear caps of 10-nm filaments (FC). These were harvested from the cells as described previously (17, 18), and the pellet of FC was washed in buffer containing 6 mM Na+/K+ phosphate (pH 7.4), 171 mM NaCl, 3 mM KCl, and 0.5 mM phenylmethylsulfonyl fluoride (Sigma). The FC were resuspended in a small volume (protein concentration about 1 mg/ml) of 6 mM Na+/K+ phosphate (pH 7.4) containing 0.1 mM phenylmethylsulfor~yl fluoride and dialyzed against 1000 vol of this buffer at 40O fr 18 hr to disperse the filaments into protofilamentous unit (i7). The opaque solution was centrifuged at 40,000 X g for 30 min and then at 250,000 X g for 1 hr. The resulting clear supernatant contained more than 75% of the total FC protein. On addition of 3 M NaCl to a final concentration of 0.17 M filaments polymerized in vitro within 6 hr at 40C. The filaments were obtained as a pellet after centrifugation at 100,000 X g for 45 min. When required, 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 "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: FC, filament caps; ORD, optical rotatory dispersion; CD, circular dichroism. t To whom all correspondence should be addressed. 6098
Cell Biology: Steinert et al.
Proc. Natl. Acad. Sc. USA 75 (1978)
I
6099
Jr Ai P. v
4L
A
I
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 0. 1.um. (D) Nine percent T/3% C sodium dodecyl sulfate-polyacrylamide gel.
ylmethylsulfonyl fluoride, dialyzed against 1000 vol of this buffer for 18 hr at 40C, clarified by centrifugation, and repolymerized on addition of 3 M NaCl to 0.17 M. Analytical Procedures. Protein was estimated by the method of Bramhall et al. (36). Polyacrylamide gel electrophoresis was performed by using a multiphasic system with 0:1% sodium dodecyl sulfate on 9% T/3% C gels (37). Electron Microscopy. Specimens were diluted to a protein concentration of about 20 Ag/ml with 6 mM Na+/K+ phosphate buffer (pH 7.4) and examined on carbon-coated ("stress-free") grids (Ladd, Burlington, VT) after negative staining with either 0.7% uranyl acetate or 1% phosphotungstic acid neutralized to pH 6.8 with KOH (38). Fibers used for x-ray diffraction were fixed in phosphate-Ixiffered glutaraldehyde, postfixed in OS04, and embedded, and ultrathin sections were stained on the grid with uranyl acetate and lead citrate (17, 18). Estimation of a-Helix Contents. Both optical rotatory dispersion (ORD) and circular dichroism (CD) were used. Samples for measurement were equilibrated at 0.1-0.2 mg/nil in 6 mM Na+/K+ phosphate buffer (pH 7.4). Measurements were made at 230C in 1-cm quartz cells with a Cary model 60 spectropolarimeter equipped with a model 6001 CD accessory. In ORD studies, the a-helix content was estimated from the mean residue rotation observed at 233 nm by using values of -1800° for 0% a-helix and -12,000° for 100% a-helix, with presumption of linear interpolation (39). Values for bo were also calculated from Moffit plots of Xo = 212 nm and were reduced to a-helix contents by using the formula (bo/-630) X 100% (40). In CD studies, the mean molar ellipticity values at 208 nm were used to calculate the a-helix contents from values of 4000° cm2dmolh' for random coil and -33,000° cm2.dmohl- for 100% a-helix (41). X-Ray Diffraction. A pellet of repolymerized filaments was used to draw fibers of 20-30 Am diameter (42). Fibers were chosen whose birefringence suggested a high degree of alignment of the filaments. A fiber was mounted in a Norelco mi-
crocamera with a specimen-to-film distance of 14-36 mm and exposed to Cu-Ka radiation (X = 1.54 A) in an atmosphere of dry helium for up to 48 hr. The 2.82-A and 3.27-A diffraction rings from the NaCl present permitted calibration of the diffraction patterns. RESULTS BHK-21 filaments can be polymerized in vitro by addition of NaCl to a final concentration of 0.17 M to a solution of solubilized 10-nm filament caps (17). On pelleting from suspension, these filaments can be disassembled with low ionic strength phosphate buffer and repolymerized on addition of NaCl to 0.17 M, with a yield of at least 50% of the starting protein. On negative staining, these filaments range in diameter from 8 to 10 nm, are about 1 gm long, and appear to have a dense-staining core throughout their length (Fig. 1 A and B). In transverse cross-section, they possess a region of diminished electron density in their centers suggestive of a tubular structure (Fig. iC); this appearance is similar to their appearance in BHK-21 cells in 3itu (43). On dissociation with sodium dodecyl sulfate followed by polyacrylamide gel electrophoresis, more than 95% of the protein associated with the repolymerized filaments appears as two bands of molecular weights about 55,000 and 54,000 (Fig. ID). Minor bands of molecular weights about 200,000 and 52,000 are also present. By ORD, solubilized FC or repolymerized filaments have a bo value of -265 and a mean residue rotation at 233 nm of -5900°. These values correspond to an a-helix content of 40-42%. By CD, their mean molar ellipticity value of -12,400°cn2.dmol-I at 208 nm corresponds to an a-helix content of 44%. Wide-angle x-ray diffraction analyses of fibers prepared from pellets of repolymerized filaments show the presence of equatorial spots at 9.7 A and meridional arcs at 5.17 A (Fig. 2). The latter reflections are significantly less than the 5.4-A spacing expected of a normal a-helix and thereby characterize this pattern as of the a type.
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 microfilaments and larger microtubules, 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.
FIG. 2.
Wide-angle
x-ray
diffraction pattern. Equatorial
spots
and meridional arcs are evident at 9.7 A and 5.17 A, respectively. The diffraction ring (at the corners) represents the 3.27-A spacing of NaCl.
DISCUSSION The present experiments demonstrate that the 10-nm filaments of BHK-21 cells, when seen in situ or after disassembly-reassembly in vitro, are long tubular structures 8-10 nm in diameter and contain 40-44% a-helix. On the basis of the x-ray diffraction pattern given by the filaments, this a-helix is arranged in a coiled-coil conformation characteristic of type proteins of the k-m-e-f class. In each a type protein so far studied, this type of structure is formed by the coiling of a-helices on either two or three adjacent subunits in the repeating structural unit of the fibrous protein. However, it remains to be determined in BHK-21 10-nm filaments whether the coiled-coil consists of two or three chains and whether the coiled-coil exists as a continuous region as in myosin (31), or is segmented (interspersed by regions of non-a-helix) as in epidermal (27) and wool (33) keratin filaments. The BHK-21 10-nm filaments share several properties with mammalian epidermal keratin filaments (42): the two have similar dimensions both in vivo and when polymerized in vitro; both appear as tubes when negatively stained or in cross-section a
(P. M. Steinert, unpublished observations); both
possess an
a
type ultrastructure; and their subunit compositions are similar in terms of net charge, molecular weight, and amino acid content, although the subunits of epidermal keratin filaments appear to be more heterogeneous. In addition, BHK-21 10-nm filaments are likewise similar to the keratin filaments of hair and wool (19) and the filaments of the inner root sheath cells of the hair follicle (44, 45), except for certain distinctive dif-
ferences in amino acid contents. Such apparent similarities to the filaments of these keratinizing tissues suggest that further structural features and properties of the 10-nm filaments may be common. The only notable difference between BHK-21 filaments and keratin filaments is their solubility properties. Whereas epidermal keratin filaments may only be polymerized low ionic strength salt solutions (42), the BHK-21 10-nm filaments dissociate under these conditions and polymerize near physiological ionic conditions.
in vitro in
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