Visualization of the large heparan sulfate proteoglycan from basement membrane

Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length means = 52 nm) and extended following negative staining (length means = 83 nm) or rotary shadowing (length means = 87 nm; 400-kDa core length means = 80 nm; P200 length means = 44 nm). Two or three glycosaminoglycan side chains (length means = 146 +/- 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.     

Characterization of human factor VIII and interaction with von Willebrand factor. An electron microscopic study

Blood coagulation factor VIII is a large glycoprotein that circulates in plasma at relative low concentration (0.1 microgram/ml). It consists of a heterogeneous mixture of a series heavy-chain peptides (90-200 kDa), each associated with a light chain of 80 kDa. To gain insight into the physical properties of the protein, we have characterized purified human factor VIII by electron microscopy and rotary shadowing. Electron microscopy of rotary shadowed factor VIII molecules showed predominantly a single globular domain structure, with a somewhat asymmetric shape, while two-domain structures were also encountered. The overall dimensions of the globular domains ranged from 4 x 6 nm to 8 x 12 nm. EDTA treatment of factor VIII reduced the overall dimensions (2.5 x 5 nm to 6 x 10 nm) while treatment with thrombin reduced the dimensions to a small extent. In complexes with von Willebrand factor, factor VIII appeared localized at the globular domains of von Willebrand factor multimers. In addition, incubation of factor VIII with Staphylococcus aureus V8 protease fragments SpII and SpIII revealed only binding to the globular domains of SpIII. In this study, the first morphological characterization of human factor VIII is presented, together with its direct localization on von Willebrand factor multimers.     

Complexes of Escherichia coli lac-repressor with non-operator DNA revealed by electron microscopy: two repressor molecules can share the same segment of DNA.

Complexes of Escherichia coli lac-repressor with non-operator DNA have been visualized in the electron microscope using high-resolution metal shadowing and negative staining. Under conditions of a high ratio of repressor to DNA, all the DNA molecules are covered by repressor molecules and the resulting complexes appear as flattened ribbons with a width of approximately 200 Å. The overall dimensions of these complexes and their substructure indicate that it is very likely that repressor molecules are tightly packed on both “sides” of the DNA helix. Thus two repressor molecules can share the same segment of non-operator DNA by binding to opposite sides of the DNA helix.     

Assembly and structure of calcium-induced thick vimentin filaments.

Using a viscometric assay and various electron microscopic procedures (negative staining, rotary shadowing, ultrathin sectioning) we have determined the influences of different kinds of ions and of ionic strength on the structures formed by assembly of soluble subunits of vimentin from bovine lens tissue or from Escherichia coli transformed with Xenopus vimentin cDNA. In contrast to the assembly of typical, i.e., 8 to 14-nm, intermediate-sized filaments (IFs) at elevated (e.g., 160 mM) concentrations of monovalent cations and at millimolar Mg2+ concentrations, filaments formed in the presence of Ca2+ ions (e.g., 5 mM) appeared at a lower rate, attained lower viscosity and were considerably thicker and shorter. The largest diameter measured was that for the recombinant amphibian protein: 24.2 +/- 8.5 nm in negative staining, 28.7 +/- 5.6 nm in sections. These thick Ca(2+)-induced filaments, however, revealed the same approximately 2 nm protofilament composition and approximately 20 nm cross-striation pattern as typical IFs, indicative of a similar molecular arrangement. The significance of this unusual structural IF protein assembly is discussed.     

The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies.

Molecules of human erythrocyte spectrin have been examined by electron microscopy after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramere were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the two polypeptide chains, band 1 (240,000 M_r) and band 2 (220,000 M_r), often appeared as two separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appears to be weak, except at both ends of the molecule where there are sites of strong binding. Tetramers are formed by the end-to-end association of two spectrin heterodimer molecules without measurable overlap, and have a mean shadowed length of 194 nm. This association to form tetramers probably involves head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode are not observed. The molecular shape of spectrin is quite distinct from that of myosin, to which it has often been likened.     

The molecular shape of dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla

The structural features of the soluble dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla were studied using negative staining and platinum shadowing electron microscopic methods. The enzyme was shown to be highly asymmetric as suggested in earlier hydrodynamic studies. The tetramer of the enzyme appeared as four subunits arranged in the shape of a planar rose with an estimated width of 15 nm. A minimum thickness of 3.0 nm for the enzyme monomer was calculated from the shadow length of unidirectionally shadowed molecules. A model composed of four oblate ellipsoid monomers in a tetrameric rose arrangement is proposed for the shape of the dopamine beta-hydroxylase molecule. Two monomers associate edge to edge to form an in-plane dimer and two dimers associate side-by-side with their respective long axes at a slight angle to form a tetramer. Theoretical calculations based on the model are consistent with previous hydrodynamic studies.     

Molecular visualization of pectin and DNA by ruthenium red

Apple fruit pectin was visualized in the electron microscope by the Kleinschmidt technique with Pt/Pd rotary shadowing, and also on benzalkonium-treated, especially thin carbon films using ruthenium red stain. Apple pectin molecules formed reticulate associations, which were partly dispersed after increasing the charge density of the molecules by enzymatic demethylation. Sycamore callus pectin molecules were visualized by the benzalkonium-ruthenium red technique as short rows of intensely electrondense dots, 3 nm across. Using the same technique, short sections of the ?X174 RF DNA double helix were visualized and the existence of the B conformation in solution directly confirmed. These observations confirm the nature of chromotropism as indicated by physical studies and provide new evidence on the staining reactions of ruthenium red.     

Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

Electron microscopy of rotary shadowed vinculin and vinculin complexes

Chicken gizzard smooth muscle vinculin, purified according to the method of Feramisco & Burridge (1980), was examined by rotary shadowing and electron microscopy. Individual vinculin molecules have two domains: a globular head with a diameter of 8.0 nm, and a tail 20 nm long. In high salt, vinculin self-associates into multimers containing two to six individual molecules. These molecules associate head to head and tail to tail, but the tail to tail association appears to be favored. Electron microscopy of the approximately 100,000 Mr major fragment of vinculin was performed. The tail region appeared to be cleaved off, making the head region less compact.     

Molecular shape and self-association of vinculin and metavinculin

Vinculin, a 130,000-dalton protein localized to adhesion plaques, and metavinculin, a 150,-000 dalton protein closely related to vinculin, have been studied using rotary shadowing and electron microscopy. Both proteins have globular head regions attached to rod-shaped tail domains. Vinculin and metavinculin also both form complexes consisting of four to six individual molecules. These multimers are formed by head-to-head as well as tail-to-tail interactions. Talin, another protein which has been localized to adhesion plaques and binds to both vinculin and metavinculin, has also been investigated using shadowing techniques. Talin is an elongated, flexible molecule in high ionic strength buffers, as shown here by rotary shadowing and negative stain electron microscopy.     

Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments

We have examined the structure of actin-binding molecules in solution and interacting with actin filaments. At physiological ionic strength, actin-binding protein has a M_r value of 540 × 10³ as determined by direct and indirect hydrodynamic measurements. It is an asymmetrical dimer composed of 270 × 10³ dalton subunits. Viewed in the electron microscope after negative staining or low angle shadowing, actin-binding protein molecules assume a broad range of conformations varying from closed circular structures to fully extended strands 162 nm in contour length. All configurations are apparently derived from the same structure which consists of two monomer chains connected end-to-end. The radius of gyration determined from the electron microscopic images was 21.3 nm in agreement with the value of 17.6 nm calculated from hydrodynamic assays. The average axial ratio from hydrodynamic measurements was 17:1, whereas fully extended dimer molecules in the electron microscope would have an axial ratio of 54:1. All of these observations indicate that actin-binding protein dimers are extremely flexible. The flexibility parameter λ (Landau &; Lifshits, 1958) for actinbinding protein is 0.18 nm^?1.As determined by sedimentation, actin-binding protein binds to actin filaments with a K_a value of 2 × 10⁶m^?1 and a capacity of one dimer to 14 actin monomers in filaments. After incubation of high concentrations (molar ratio to actin ≥ 1:10) of actin-binding protein with actin filaments, long filament bundles are visible in the electron microscope. Under these conditions, actin-binding protein molecules decorate the actin filaments in the bundles at regular 40 nm intervals or once every 15 monomers, approximately equivalent to the binding capacity measured by sedimentation. Low concentrations of actin-binding protein (molar ratio to actin ≥ 1:50) which promote the gelation of actin filaments in solution, did not detectably alter the isotropy of the actin filaments. Direct visualization of actinbinding protein molecules between actin filaments in the electron microscope showed that dimers are sufficient for crossbridging of actin filaments and that actinbinding protein dimers are bipolar, composed of monomers connected head-to-head and having actin-binding sites located on the free tails.We conclude that actin-binding protein is a dimer at physiological ionic strength. Each dimer has two actin filament binding sites and is therefore sufficient to gel actin filaments in solution. The length and flexibility of the actin-binding protein subunits render this molecule structurally suited for the crosslinking of large helical filaments into isotropic networks.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin

The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.     

Reversible embedment cytochemistry (REC): a versatile method for the ultrastructural analysis and affinity labeling of tissue sections

Reversible embedment cytochemistry (REC) is a new method for revealing cellular ultrastructure and for improving access of intracellular targets to macromolecular affinity labels. Fully polymerized polymethylmethacrylate was dissolved in dichloromethane and infiltrated into fixed tissue-culture cells and tissues. After evaporation of the solvent, samples were left in hard plastic. Samples were thus embedded without exposure to chemical polymerization reactions that might damage tissue ultrastructure or antigenicity. Glass or diamond knives fitted with water troughs were used to cut sections 30-1000 nm thick. Since polymethylmethacrylate is composed of linear polymers that are not covalently crosslinked, the plastic was easily extracted from the sections by immersion in solvent. Subsequently, various preparative methods, including negative staining, critical point-drying, and platinum-carbon rotary shadowing, were used to provide detailed images of well-preserved cell structure for conventional and high-voltage transmission electron microscopy. Fluorescein-conjugated affinity labels were used to obtain subcellular distributions of target molecules in semi-thick sections of cultured cells and tissues for light microscopy. Colloidal gold-labeled antibodies were used to localize microtubules in sections of cultured cells by electron microscopy. REC is a versatile method that should find wide application in many studies of cellular function.     

Melting of myosin and tropomyosin: electron microscopic observations

A method was devised to maintain a very low angle (2-3 degrees) during the metal casting of specimens for electron microscopy. With this modified rotary shadowing procedure the melting of myosin and tropomyosin (TM) was investigated. When protein solutions were sprayed on mica sheets and then heated to melt alpha-helices, myosin molecules did not show any sign of chain separation but appeared to have collapsed into loose clumps. A few molecules showed separation of the two chains at the light meromyosin-heavy meromyosin hinge region. Heating myosin in bulk solution at 65 degrees C before spraying caused extensive fusing of the myosin heads. In contrast, in the case of TM, separation of the chains appeared to occur at temperatures at which the unfolding of alpha-helices had been shown by circular dichroism. Dissolution of TM and myosin in 0.5% SDS followed by 150-fold dilution led to single chain species. This method capable of detecting single chain peptides of melting TM whose thickness is of the order of 1 nm may be applicable to the study of the structure of proteins previously not considered possible.     

An analysis by rotary shadowing of the structure of the mammalian vitreous humor and zonular apparatus

An electron microscopic analysis of human and bovine vitreous humor after rotary shadowing showed the presence of both collagen fibrils and an extensive loose network of hyaluronan molecules. No interaction between the collagen fibrils and the hyaluronan molecules was observed under the conditions used for rotary shadowing. Periodic "struts" were present on the surface of the collagen fibrils. These struts showed an organization the same as that previously observed for type IX collagen on the surface of collagen fibrils from chicken cartilage and vitreous. However, the knob of the noncollagenous NC4 domain of cartilage type IX collagen was not observed at the ends of the struts in a manner identical to that of chicken vitreous humor. Zonular fibrils were dissected out from bovine eyes and shown by rotary shadowing to contain a beaded fibril which is similar in morphology to the "elastin-associated" microfibrils of many connective tissues. Experiments in which the zonular fibrils were stretched and fixed prior to rotary shadowing showed that the distance between each bead is variable and can be accounted for by the bowing out of overlapping filaments which connect each bead.     

AN ultrastructural study of cross-bridge arrangement in the frog thigh muscle thick filament.

We have developed thick filament isolation methods that preserve the relaxed cross-bridge order of frog thick filaments such that the filaments can be analyzed by the convergent techniques of electron microscopy, optical diffraction, and computer image analysis. Images of the filaments shadowed by using either unidirectional shadowing or rotary shadowing show a series of subunits arranged along a series of right-handed near-helical strands that occur every 43 nm axially along the filament arms. Optical filtrations of images of these shadowed filaments show 4-5 subunits per half-turn of the strands, consistent with a three-stranded arrangement of the cross-bridges, thus supporting our earlier results from negative staining and computer-image analysis. The optical diffraction patterns of the shadowed filaments show a departure from the pattern expected for helical symmetry consistent with the presence of cylindrical symmetry and a departure of the cross-bridges from helical symmetry. We also describe a modified negative staining procedure that gives improved delineation of the cross-bridge arrangement. From analysis of micrographs of these negatively stained filament tilted about their long axes, we have computed a preliminary three-dimensional reconstruction of the filament that clearly confirms the three-stranded arrangement of the myosin heads.     

Corneal and scleral type I collagens: analyses of physical properties and molecular flexibility

The physical properties of type I collagen were studied by electron microscopy of rotary shadowed collagen molecules and laser light scattering techniques. The physical properties, molecular structure and flexibility of type I collagen molecules from two structurally and functionally different connective tissues, cornea and sclera, were similar when measured in HCl, pH 2.0. The molecular weights were 328 and 298 × 10² for corneal and scleral type I collagen, respectively, while the values of T_M were 33.7°C for both preparations. These values were in agreement with those obtained for other type I collagens. The higher level of glycosylation in corneal versus scleral type I collagen did not significantly modify the physical properties of type I collagen in acid solution or the charge distribution along the molecule as determined from the positively stained SLS banding patterns. Our morphological studies indicated that the collagen molecule, although relatively flexible based on electron microscopy, behaved as a long thin rod in solution. The mean end-to-end distances measured from electron micrographs were 253 and 256 nm for corneal and cler type I collagen, respectively, while the molecular contour lengths were 298 and 305 nm. The translational diffusion coefficients (0.849 and 0.857 × 10^?7cm²s^?1) were consistent with the contour lengths while the reported values in the literature for the rotational diffusion coefficient of type I collagen were consistent with the end-to-end distances. The intermediate value for molecular length obtained from the particle scattering factor (277 nm) reflects contributions from all possible molecular configurations.     

Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

Shadowing of elongated helical molecules (myosin, tropomyosin, collagen, and DNA) yields regular molecule-dependent heavy metal grain patterns

Myosin and other alpha-helical molecules (tropomyosin, collagen) can now directly be adsorbed on EM support films, washed, air-dried, or frozen and freeze-dried. Using this method, the molecules were rotary or unidirectionally shadowed with different heavy metals (Pt/C, Ta/W, Ag) or with C alone. After shadowing at low elevation angles with Ta/W or Ag, myosin, tropomyosin, collagen, and DNA showed strikingly regular patterns of either single or coalesced heavy metal grains (bands) along their entire lengths. Even after shadowing with C alone, repetitive, granular accumulations or bands of C were found along the molecules. The different heavy metals and C displayed distinctive banding patterns on the molecules examined, all of which are characterized by different surface charge periodicities and pitch values. The patterns were quantified on the basis of the distances between grains or bands. Two most frequently measured distances between bands were found after shadowing with heavy metals. After shadowing with Ag the prevalent distances between grains were about twice as large as those after Ta/W shadowing. By evaporating a thin layer of carbon on the molecules before shadowing with heavy metals or by evaporating C alone (with no heavy metal) at 6 degrees, one of these two most prevalent distances between bands was attenuated or disappeared. It was demonstrated that the remaining most frequently measured distances between grains seemed to be related to relief periodicities, to the pitch of the double-coiled (myosin, tropomyosin) and triple-coiled alpha-helices (collagen) and fractions thereof. The attenuated distances between grains agreed very well with distances of periodic surface charges on the molecules examined. The investigation of the grain or band patterns showed that their characteristics appearance was molecule-dependent and caused both by periodic chemical (repeats of positive and negative surface charges) and periodic structural features (coiling of the helical strands). The examination confirmed the existence of periodic positive and negative surface charges along the myosin rod and suggested a value of about 17.0 nm for the hitherto undetermined pitch of the double-coiled myosin rod.