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Keratin Filaments Have Similar Structures (Electron Microscopy/Spectropolarimetry/Wide-Angle X-Ray Diffraction) PETER M

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Keratin Filaments Have Similar Structures (Electron Microscopy/Spectropolarimetry/Wide-Angle X-Ray Diffraction) PETER M 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 structure of keratin filaments from both hair (and wool) and (BHK-21) cells, when examined either in the form of native epidermis is now well advanced (26, 27). Thus consideration ilanent caps or polymerized in vitro, are long tubes of protein 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 proteins 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 (myosin)-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), actin-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 neurofilaments 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 desmin (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 vertebrate 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 keratinocytes, 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 amino acid 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 Jr A i I 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 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 staining 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.
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