Conformity and diversity in the structures of intermediate filaments.
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Animals↗
Biomedical subjects
A C Steven
Publications and source records attributed to A C Steven.
Structural and functional implications of amino acid sequences of keratin intermediate filament subunits.
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Amino Acid Sequence↗
Purification and characterization of fimbriae isolated from Bordetella pertussis.
Fimbriae were detached from Bordetella pertussis by mechanical shearing and purified by successive precipitations with ammonium sulfate, phosphate buffer (pH 6.0), and magnesium chloride. In each of these purification steps, the fimbriae aggregated into bundles as seen by electron microscopy. These aggregates could be disaggregated at pH 9.5. By electron microscopy, the purified fimbriae appeared as long filaments with a diameter of 5 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed a single protein subunit with a molecular weight of 22,000. The purified fimbriae did not have hemagglutinating activity when assayed with several types of erythrocytes, and they were antigenically, chemically, and structurally distinct from the filamentous hemagglutinin of B. pertussis. The purified fimbriae were also identified as serotype 2 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.2.4, 1.2.3, or 1.2.3.6 but did not agglutinate those serotyped as 1.3.6.
Agglutination↗
Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.
Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.
Capsid↗
Purification of serotype 2 fimbriae of Bordetella pertussis and their identification as a mouse protective antigen.
Fimbriae were removed from Bordetella pertussis by mechanical shearing and purified by successive precipitations with ammonium sulfate, pH 6.0 phosphate buffer, and magnesium chloride. Electron microscopy showed the purified fimbriae to be long filamentous structures (5 nm in diameter) which aggregated into bundles at pH 6.0. Sodium dodecyl sulfate gel electrophoresis of the purified fimbriae gave a single protein subunit with a molecular weight of 22,000. The purified fimbriae did not have hemagglutinating activity when assayed with a variety of erythrocytes and were shown to be antigenically and structurally distinct from the filamentous hemagglutinin of B. pertussis. The purified fimbriae were identified as serotype 2 agglutinogens as antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.2.4, 1.2.3, or 1.2.3.6, but did not agglutinate serotypes 1.3.6. Immunization of mice with the purified fimbriae (14 micrograms) protected them from a lethal respiratory infection with strain 18323 (agglutinogen serotype 1.2.3.4.6) or with strain 432 (serotype 1.3.6). Immunization of mice with purified fimbriae containing 0.02% LPF also protected them from a lethal intracerebral infection with 18323. The ED50 was about 13 micrograms. Fimbriae containing less than 0.005% LPF did not protect mice from intracerebral challenge.
Animals↗
Structural studies of muscle during force development in various states.
Structural studies concerned with force generating mechanisms in striated muscle fibers in different states are described. The first study deals with fibers in the "rigor" state, where ATP is absent and all the myosin heads form cross-bridges with the actin-containing filaments. In this state large axial forces are developed when ionic strength is reduced to very low levels. At the same time, the fiber expands radially, as indicated by both X-ray diffraction and light microscopy. Comparison of the latter two measurements indicates that force is developed in part because of differences in the lateral expandability of different parts of the sarcomere. Thus, under these conditions, force appears to be modulated by factors that operate at the filament rather than the cross-bridge level. The second study deals with the location of the myosin heads in the relaxed state, and the mass movement that takes place when the fiber is physiologically activated. By using the intense X-ray source at the Stanford Synchrotron Radiation Laboratory, five equatorial reflections were recorded for both the relaxed and the activated state, and a spatial resolution of 100 A was obtained. Analysis of the data indicates that (1) in the resting state, myosin heads protrude out from the thick filaments and extend toward the thin filaments, and (2) upon activation, significant loss of mass occurs only in the region peripheral to the thick filament backbone through a movement that has a pronounced azimuthal component. The latter movement can be taken as the overall shift in myosin subfragment 1 during the cross-bridge cycle.
Actins↗
Diffraction patterns from stained and unstained helices: consistency or contradiction?
Addressing the issue of how best to integrate negative staining electron microscopy with X-ray fiber diffraction of macromolecular helices, we investigated staining properties of helical models using computational simulations. We compared diffraction patterns of stained and unstained representations of F-actin, myosin S1-decorated actin, and microtubules, to test the reliability of selecting well preserved specimens as those whose optical diffraction patterns most closely match the X-ray patterns of hydrated fibers. We conclude that if the stain layer is considerably thicker than the outer diameter of the specimen, this criterion is likely to be reliable. However, specimens in such thick stain layers may be resolution-limited by dynamic scattering effects such as beam-broadening. With relatively thin staining (the more practically relevant situation) "edge effects" in the stain distribution result in differences between diffraction patterns of stained and unstained specimens. Diffraction pattern changes due to molecular distortions such as shrinkage or flattening were similarly modeled. Since differences between diffraction patterns of stained and unstained helices may be due to either "edge effects" or molecular distortions, it does not appear possible a priori to distinguish between these effects. Comparison of experimental data for the (2.3 nm)-1 layer-line of tobacco mosaic virus (as model system) reveals major differences between the X-ray diffraction pattern of hydrated sols and the Fourier transform of HREM images of negatively stained specimens.
Microscopy, Electron↗
The complete cDNA and deduced amino acid sequence of a type II mouse epidermal keratin of 60,000 Da: analysis of sequence differences between type I and type II keratins.
We present the complete nucleotide and deduced amino acid sequences of a mouse epidermal keratin subunit of 60,000 Da. The keratin possesses a central alpha-helical domain of four tracts (termed 1A, 1B, 2A, and 2B) that can form coiled-coils, interspersed by short linker sequences, and has non-alpha-helical terminal domains. This pattern of secondary structure is emerging as common to all intermediate filament subunits. The alpha-helical sequences conform to the type II class of keratins. Accordingly, this is the first type II keratin for which complete sequence information is available, and thus it facilitates elucidation of the fundamental distinctions between type I and type II keratins. It has been observed that type I keratins are acidic and type II keratins are neutral--basic in charge. We suggest that the basis for this empirical correlation between type and charge resides in the respective net charges of the 1A and 2B tracts. Calculations on interchain interactions between charged residues in the alpha-helical domains indicate that this keratin prefers to participate in dimers according to an in-register parallel arrangement. The terminal domains of this keratin possess characteristic glycine-rich sequences, and the carboxyl-terminal domain is highly homologous to that of a human epidermal keratin of 56,000 Da. According to the hypothesis that end-domains are located on the periphery of keratin filaments, we conclude that the corresponding mouse and human keratins are closely related, both structurally and functionally.
Amino Acid Sequence↗
Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs.
A procedure has been developed for direct determination of radial distributions of density in filamentous and spheroidal particles by analyzing dark-field scanning transmission electron micrographs of unstained freeze-dried specimens. Unlike electron microscopic methods based on staining or shadowing with heavy atoms, this approach can be used to probe the internal structure of macromolecular complexes. As an experimental proving ground, we have applied the procedure to tobacco mosaic virus (TMV) and to RNA-free helical polymers of TMV coat protein. Both structures are found to project outermost diameters of 17.6 +/- 0.4 nm, to have empty axial holes approximately equal to 3.5 nm in diameter, and to have density peaks at radii of 2.5 +/- 0.5 and 6.7 +/- 0.3 nm. Thus visualized, the only significant difference between them is the presence in the virion of an additional density peak at 4.1 +/- 0.5 nm contributed by its internalized RNA molecule. We have also used the procedure to monitor the structural expression of radiation damage in the low electron dose regime prior to the onset of significant mass loss. Changes in the radial density profiles are detected at average doses as low as approximately equal to 400 electrons per nm2: the trend is for the internal structure of these particles to fuse toward a state of uniform density, although the values of their outermost diameters remain unaffected.
Densitometry↗
Detection of membrane cholesterol by filipin in isolated rat liver coated vesicles is dependent upon removal of the clathrin coat.
We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin. Failure of this reagent to elicit its typical response for a cholesterol-containing membrane, i.e., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A. Perrelet, P. Vassalli, and L. Orci, 1979, Proc. Natl. Acad. Sci. USA., 76:6391-6395) that cholesterol is specifically excluded from the plasma membrane domains associated with coated pit regions. The present electron microscopic results showed that although the response of coated vesicle membranes to filipin was also negative, uncoated vesicles whose clathrin coats had been removed in vitro exhibited a strong filipin-positive response. Quantitated biochemically, the cholesterol-to-phospholipid ratio of the coated vesicles was found to be indistinguishable from that of control preparations of plasma membranes isolated from rat liver. Taken together, the results indicate that the filipin-negative response of coated vesicles (and probably also that of coated pits) is due not to abnormally low cholesterol content, but rather to the stabilizing influence of their enveloping clathrin coats which inhibit the characteristic structural expression of the filipin-cholesterol complexes.
Animals↗
The distribution of mass in heteropolymer intermediate filaments assembled in vitro. Stem analysis of vimentin/desmin and bovine epidermal keratin.
We have studied the distribution of mass in several types of intermediate filaments (IF) assembled in vitro, by analyzing scanning transmission electron micrographs (STEM) of unstained specimens imaged in dark-field mode. Bovine epidermal keratin IF, which are obligate heteropolymers, were thus characterized and compared with facultative heteropolymers of vimentin and desmin and with earlier observations of homopolymer vimentin IF. The major components of each type of IF have linear densities of 37 kilodaltons/nm. Minor polymorphic variants are also present in each case, with linear densities of approximately 25 and 48 kilodaltons/nm, respectively. In view of the known subunit masses, these results are consistent with the proposition that at least three IF types (bovine epidermal keratin, vimentin, and desmin) are structurally homologous. For each type of IF studied, measurements taken near the ends of long IF or from short IF tend to have lower densities, characteristic of the least dense polymorphic variant. This form may represent a precursor "minimal core" structure in the sense of a minimal aggregate of protofilaments sufficiently stable to permit elongation. In the course of in vitro assembly, this form would be maturable to the normal IF structure at 37 kilodaltons/nm by the accretion of further protein. We also find the projected widths of these IF as measured from the STEM images to be significantly greater than the corresponding diameters determined in previous studies by conventional electron microscopy. Whereas the conventional techniques have routinely yielded diameters of 7-11 nm for IF contrasted by heavy metal staining, the STEM images of unstained IF provide estimates of 15 +/- 1 nm for the outer diameter of the major density class for each IF type studied and average values of 13.8 and 16.1 nm for the less dense and more dense variants, respectively.
Animals↗
Complete amino acid sequence of a mouse epidermal keratin subunit and implications for the structure of intermediate filaments.
We have determined the complete primary structure of an intermediate filament subunit, the 59,000 molecular weight subunit of mouse epidermal keratin, from the nucleotide sequence of cDNA clones. The central portion of the sequence forms extended tracts of a coiled-coil alpha-helical conformation. This is flanked at both termini by similar non-alpha-helical sequences that are extremely rich in glycine residues, frequently configured in tandem peptide repeats. Limited chymotryptic digestion of keratin filaments containing this protein suggests a structural organization whereby the terminal glycine-rich sequences protrude from a conserved core structure into which the coiled-coil alpha-helical segments are packed.
Amino Acid Sequence↗
Molecular architecture of bacteriophage T7 capsid.
To determine the capsid structure of bacteriophage T7, we have investigated polycapsids, tubular capsid-related structures isolated from lysates of the T7 mutant am16. Biochemical analysis shows polycapsids to be composed of gp10, the major structural protein of the wild-type capsid. The conformational state of gp10 in polycapsids is indistinguishable from that in the mature virus capsid by the criteria of surface charge, buoyant density, and insensitivity to proteolysis by trypsin. Optical diffraction of electron micrographs of negatively stained polycapsids reveals a hexagonal surface lattice of periodicity 12.6 +/- 0.2 nm and is used to analyze the distribution of cylindrical foldings of this lattice into polycapsids (polymorphic variation). These foldings are found to be related to that of the capsid proper through the intrinsic curvature of gp10, each folding having a set of lattice lines whose radius of curvature is close to 29 nm. The fine structure of this surface lattice has been elucidated by digital image processing of electron micrographs. The capsomer is shown unequivocally to be a hexamer of characteristic morphology. By collating these results with earlier observations, we conclude that the structure of the normal T7 capsid is an orthodox icosahedron of triangulation class T = 7, composed of 60 hexamers and 12 pentamers.
Capsid↗
Mass distributions of coated vesicles isolated from liver and brain: analysis by scanning transmission electron microscopy.
Populations of coated vesicles purified from bovine brain (BCV) and from rat liver (LCV) have been characterized with respect to the parameters of mass and diameter by analysis of scanning transmission electron micrographs of unstained specimens. Coated vesicles from both sources are heterogeneous, particularly in their masses. The respective distributions, compiled from mass measurements of many individual particles, are complex and markedly different. BCV range from 20 Mdaltons to approximately 100 Mdaltons with a weighted average of 35 Mdaltons: most BCV (80%) lie between 20 and 40 Mdaltons, including peaks at approximately 26 Mdaltons and at approximately 34 Mdaltons. In contrast, LCV masses tend to be substantially higher, ranging from 20 to 220 Mdaltons with a weighted average of 66 Mdaltons. There is a prominent subpopulation at approximately 35 Mdaltons, and 59% of all LCV belong to a broad peak between 50 and 120 Mdaltons. The Kolmogorov-Smirnov distribution-free test was used to affirm the statistical reproducibility of these isolates. BCV diameters vary from 50 to 90 nm, and those of LCV from 50 to 150 nm. Both protein compositions, determined by SDS PAGE, are dominated by clathrin and they are generally similar, except that corresponding secondary bands, notably the clathrin-associated light chains, appear to have lower molecular weights in the case of LCV. From consideration of the joint mass-diameter distribution, it is apparent that coated vesicles of a given diameter vary considerably in mass and that this variation is due primarily to widely differing amounts of material enclosed within the clathrin coat.
Animals↗
Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments.
We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers averaging 33 and 35 kdalton/nm, respectively, which probably represent mature IF. Taken together with earlier results on IF of other subclasses, these results indicate that the average linear density of IF scales according to the average mass of their constituent subunits, both for "minimal form" and for mature IF. As underlying mechanism for this homology, we propose that the fundamental building-blocks of all these IF contain a common structural element whose packing within the various IF is likewise conserved and which specifies the overall structure. The variable amounts of mass in the nonconserved moieties account for the observed proportionality. This scheme fits with amino acid sequence data for several IF subunits that have revealed, as a likely candidate for the common element, an essentially conserved alpha-helical domain, contrasting with the highly variable sequences of their non-alpha-helical terminal domains.
Animals↗
Structural features of epidermal keratin filaments reassembled in vitro.
We have studied the structure of epidermal keratin filaments polymerized in vitro, addressing two different levels of organization. First, we have determined the amino acid sequence of a mouse epidermal keratin subunit from the nucleotide sequence of a cDNA clone. The subunit contains a large central region, representing about 50 percent, whose sequence strongly suggests that it assumes a coiled-coil alpha-helical conformation. This is flanked on the amino and carboxyl terminals by long glycine-rich sequences. Second, we have used scanning transmission electron microscopy to study the structure of frozen, unstained filaments. Analyses of such images provides information on the mass per unit length and on the distribution of mass within the filament. These data impose rigorous constraints on possible models for the packing of protofilaments within the filament. Epidermal keratin filaments assembled in vitro are polymorphic; however, the majority of bovine filaments weigh about 37 kD/nm, but most human filaments have masses of only about 27 kD/nm. The filament width is at least 15 nm, substantially more than the generally accepted value of 8 to 10 nm, owing to the existence of low-density mass at the periphery that has not been visualized by conventional microscopic methods. We currently postulate that the alpha-helical regions of the subunits comprise the structural core or backbone of the filament from which at least some of the glycine-rich sequences protrude.
Amino Acid Sequence↗
Structure of fibroblastic intermediate filaments: analysis of scanning transmission electron microscopy.
The structure of fibroblastic intermediate filaments from Chinese hamster ovary cells has been investigated by scanning transmission electron microscopy. Freshly extracted (native) filaments were compared with filaments reassembled in vitro from purified decamin. From digital micrographs of unstained specimens, direct measurements of linear mass density were performed on many individual filaments. Native filaments beyond a certain minimal length constitute a homogeneous population, averaging 38 +/- 4 kilodaltons (kDal)/nm. A minor but distinct polymorphic variant (23 +/- 4 kDal/nm) was also present as very short filaments or end-segments; these may represent breakdown products or assembly intermediates. Analysis of reassembled filaments demonstrates that the in vitro assembly reaction is--in the main--faithful, although the distribution of their mass measurements is appreciably broader than that of the native data. In addition to the predominant type at 37 +/- 4 kDal/nm and a minor component at 26 +/- 4 kDal/nm, small amounts of a third, more massive, polymorphic variant at 52 +/- 5 kDal/nm were also present. Micrographs of negatively stained specimens clearly demonstrate that the filaments are composed of bundles of protofilaments--each 2-3 nm in diameter--and also reveal an axial periodicity of about 46 nm. The implications of these findings are discussed for three classes of model previously proposed for the structure of intermediate filaments.
Animals↗
Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate.
The mechanism of contrast enhancement of protein molecules by negative staining with uranyl acetate has been investigated by analysing electron micrographs of microcrystals of the human immunoglobulin Dob. Digitally filtered micrographs were compared systematically with idealized reference images which were constructed computationally, starting from knowledge of the primary sequence and three-dimensional crystal structure of this IgG molecule. By separately modelling negative staining as bulk exclusion of heavy metals, and positive staining as the specific decoration of charged amino acid residues, and then combining these simulated images, we were able to assess quantitatively the amount of positive staining present in micrographs of ostensibly 'negatively stained' proteins. At a resolution of 2 nm, we find that the experimental images do indeed exhibit predominantly negative staining, the best matches being obtained by simulations which also include a minor contribution (10-40%) of positive staining. We have also compared two independent measures for the significant resolution present in images of periodic biological specimens: (i) the outermost visible orders of optical diffraction patterns, and (ii) the band-limited resolutions of the idealized simulations when they most closely match the experimental images. These criteria observe close correspondence, thus vindicating the traditional practice of inferring resolution from the optical diffraction spectra of indirectly represented (stained) objects.
Computers↗