10 nm filaments in normal and transformed cells.

An antibody was raised against an electrophoretically homogeneous protein from cultured fibroblasts and shown to be directed against 10 nm filaments. The antiserum did not stain microtubules or actin microfilaments. The distribution of 10 nm filaments in normal cells was studied during growth, spreading, locomotion, mitosis, and after treatment with colchicine and cytochalasin B. The 58,000 dalton subunit protein is apparently all polymerized in the filaments which are insoluble in nonionic detergent. The distribution of 10 nm filaments is altered by colchicine treatments which disrupt microtubules. The organization of 10 nm filaments is altered in transformed cells.     

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.     

Fine structure of bovine lens epithelial cells in vitro in relation to modifications induced by a retinal extract (EDGF)

Cultured bovine lens epithelial cells are polygonal in shape. In confluent and multilayer cultures they exhibit elaborate arrays of 6 nm filaments, bundles of intermediate-sized filaments, and a fibrous meshwork of subcellular and intercellular material. Cells grown in the presence of a retinal extract (RE) have a higher growth rate, and are smaller and more regular in shape. In them the 6 nm filaments are mostly aligned in sheets, the intermediate-sized filaments form a fine network, and the cells are closely apposed to the plastic substratum. Some homogeneous material is formed intercellularly in older cultures. Cellular elongation, induced in the former cultures by the addition of RE, is accompanied by an alignment of cytoskeletal elements, including microtubules, parallel to the long axis. Other structural features are similar in all cell types. The response to RE is discussed in terms of shape modulations associated with the restricted expression of structural characteristics acquired in vitro.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cytoskeletal function

Cow Purkinje fibers contain a population of free cytoplasmic filaments which consistently differ in ultrastructural appearance from actin and myosin filaments, irrespective of preparation technique. The fixation and staining techniques, however, influenced the filament diameter, which was found to be 7.4--9.5 nm for filaments in plastic-embedded material, and 7.0 nm in cryo-sectioned material, thus intermediate as compared to actin and myosin filaments. Cross-sectional profiles suggested that the intermediate-sized filaments are composed of four subfilaments. To provide a basis for further biochemical investigations on the filaments, extraction procedures were carried out to remove other cell organelles. Electron microscopy showed that undulating bundles of intermediate filaments converging towards desmosomes still remained, after the extractions, together with Z-disk material. In spite of the extensive extraction, the shape of the individual cells and the assemblies of cell bundles remained intact. This confirms that the intermediate filaments of cow Purkinje fibers together with desmosomes do in fact have a cytoskeletal function. On account of (a) the cytoskeletal function of the filaments, (b) the similarities to the smooth muscle "100-A filament" protein subunit skeletin, and (c) the inadequate and confusing existing terminology, we suggest that the filaments be named "skeletin filaments."     

Structure and development of the surface coat of erythrocytic merozoites of Plasmodium knowlesi

Summary The surface of extracellular merozoites of P. knowlesi is covered with a coat 15–20 nm thick, made up of clusters of filaments standing erect on the plasma membrane. Filaments have stems 2 nm thick, the peripheral ends of which are complex, branching or ending in long trailing threads. Coat filaments occur on the surface of the parasite in regular rows at an early schizont stage, and persist until well after merozoite release. They are sensitive to trypsin and papain, and bind ethanolic phosphotungstate, indicating a proteinaceous nature. They are also removed by exposure to phosphate-buffered saline. Filaments bear negative charges, binding cationised ferritin throughout the depth of the coat and staining with ruthenium red. They cover the whole merozoite surface and mediate intercellular adhesion at distances of 15–150 nm, membrane to membrane. It is suggested that these filaments correspond to a major merozoite surface protein, and are important in the initial capture of red cells.     

THE FINE STRUCTURE OF THE SHELL MUSCLE OF PATELLID PROSOBRANCH LIMPETS

The structure of the shell muscle of eleven species of patellidlimpet is described from light and transmission electron microscopestudies. Although the muscle has many structural characteristicstypical of molluscan smooth muscle, it also has a number ofunusual features. At the electron microscope level two myofibretypes are distinguishable. Type I cells, present in all species,contain conventional contractile apparatus in the form of thickand thin filaments. Thick filaments contain paramyosin and varyin diameter between 20—180 nm. An axial striation witha repeat of 14.2 nm is calculated from optically diffractedmicrographs of isolated thick filaments. Transverse sectionsof thick filaments reveal bands from which the transverse repeatof the paramyosin crystal lattice is calculated. Type II myofibres,which are present in five species, contain a novel arrangementof thin filaments with electron-dense regions at intervals of80–150 nm. The striated thin filaments are similar inappearance to the microfilament bundles and stress-fibres ofnon-muscle cells. They also have similarities to the leptomericorganelles of some vertebrate muscle tissues. Associated withthe muscle is an unusually large amount of collagen which hasa periodicity of 62 nm calculated from optical diffraction patternsof isolated collagen fibrils. (Received 3 July 1989; accepted 12 October 1989)     

Reconstitution of intermediate filaments from a higher plant.

Immunological studies have shown that plants contain intermediate-filament antigens, but it is not known whether these proteins are capable in themselves of forming filaments. To address this problem, a detergent-resistant and high-salt-insoluble fraction from carrot (Daucus carota L.) suspension cells was solubilized with 9 M-urea and then subjected to a two-step dialysis procedure, devised for the reconstitution of animal intermediate filaments. This induced the self-assembly of 10 nm filaments and large bundles of filaments. The predominant components of reconstituted material were polypeptides with apparent molecular masses between 58 and 62 kDa. These polypeptides immunoblotted with two monoclonal antibodies known to show broad cross-reactivity with intermediate filaments across the phylogenetic spectrum. This establishes that the antigens are able to self-assemble into intermediate-sized filaments.     

Filamentous structures in adherent Mycoplasma pneumoniae cells treated with nonionic detergents

Mycoplasma pneumoniae cells adhering to glass or Parlodion-coated grids were extracted with Triton X-100. The extracted cells showed a cytoskeleton consisting of a rodlike tip structure and a filamentous network in the cytoplasm. The tip structure was up to 300 nm long and approximately 40 nm wide ending at the distal end in a bleb-like structure, and seemed to consist of filaments arranged in parallel, 4.8 +/- 0.5 nm wide. In the cytoplasm the filaments formed an irregular lattice. Similar filaments were found in platinum replicated critical- point dried extracted cells. An actinlike nature of the filaments is suggested by some of their properties, but the degree of homology with respect to eucaryotic actin is still unknown. The filaments were sensitive to protease treatment but stable in high molar KCl solutions. They were apparently destroyed by incubation in high molar KI solution, leaving only some parts of the tip structure. Formaldehyde-fixed M. pneumoniae cells treated with Triton X-100 bound rhodamine-labeled phalloidin specifically. Furthermore, they could be stained with antiactin antibodies. Binding of myosin subfragment 1 to the filaments was not observed.     

The Occurrence and Some Properties of a New Filamentous Component of Plant Extracts

The electron microscopic image and some properties of a filamentouscomponent of plant extracts are described. The filaments are3·5 nm in diameter, non-rigid and present a beaded appearancewhen positively stained with uranyl and lead salts. They arepresent in the microsomal supernatant fractions of a varietyof plants, and can be obtained in extracts of fresh tissuesor of acetone powder preparations. Specific tissues, successfullyextracted for filaments, include phloem, xylem and corticalparenchyma. They are not present in extracts of cell wall preparationsor of hair cells of cotton bolls. An outstanding characteristicof these filaments is their great stability to a wide varietyof treatments which includes variation in temperature, ionicenvironment, pH and the presence of urea, thiol reagents orthe detergent Nonidet. Lateral aggregation of the filamentsis evident below pH 3·0 and above pH 8·0. Althoughcellulase was the only enzyme of those tested which digestedthe filaments, it is unlikely, for several reasons, that thefilaments are a form of pure cellulose. Hydroxyproline was presentin all filament fractions after partial purification by variousmethods. The filaments are discussed in relation to known fibrillarcomponents of plant cells.     

Association of fibronectin with the microfibrils of connective tissue

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Microfilament-undercoated plasmalemmal infoldings in a human clear-cell sarcoma

Unusual structures often found in the cytoplasm of tumor cells in a clear-cell sarcoma appeared as multilayered, concentric, oval, spiral, parallel arrays of cisternae in various planes of section. It was demonstrated that the cisternal membrane and cavity were continuous with plasmalemmas of tumor cells and the extracellular space, respectively, suggesting that the structures were formed by the intracytoplasmic infoldings of plasmalemmas. Another characteristic found in the structures was orderly microfilaments with an average diameter of 6.5 nm which were placed between the confronting plasmalemmas in the infoldings. The filaments which underlay the infolded plasmalemmas ran parallel to each other along the cytoplasmic surfaces of plasmalemmas approximately 15 nm apart. The regularly arranged filaments were found in the infolded plasmalemmas, but not beneath any other area of plasmalemmas. The short ends of long filaments appeared to bend toward the inner surfaces of plasmalemma and to be directly connected with the surface proper. These results show that the filaments may be closely associated with the plasmalemmal infoldings and included as the same category of plasmalemmal undercoat. Additionally, the biological significance of the structures is discussed.     

Electron microscopic analysis of lymphocyte fibrillar elements during the redistribution of surface receptors

Filaments 5 nm thick, located throughout the cytoplasm mainly along the surface, are observed in intact lymphocytes. In the control glycerinized lymphocytes, besides the above filaments aggregations of filaments nearly 3 nm in diameter were found. After the treatment of cells by antimurine serum or ferritin-conjugated concanavalin A, some fibrillar structures are observed mainly in the cap region in the form of filaments 5-6 nm of thickness, radially directed towards the plasma membrane. After glycerinization, three types of filaments are observed being, respectively, near 3, 5-6 and almost 8 nm in diameter. Two latter types of filaments are decorated by S1-myosine fragments which indicates their actine nature. Differences in the character and distribution of myofibrils in the cytoplasm of intact cells and cells with caps may witness in favour of their involvement in the processes associated with redistribution of surface receptors.     

An ultrastructural investigation of muscle attachment in the opercular filament of a polychaete annelid

The ultrastructure of peduncle muscle attachment to the cuticular flange in the opercular filament of the serpulid Pomatoceros lamarckii Quatrefages is described. The cuticular flange is composed of layers of orthogonally arranged fibres. Specialized epithelial cells (tendon cells) and a collagenous matrix intervene between the peduncle muscles and the cuticular flange. The tendon cells are characterized by hemidesmosomes at both apical and basal ends, connected by thick bundles of tonofilaments. Apically long specialized microvilli from the tendon cells penetrate the cavities in the orthogonally arranged layers of fibres of the cuticular flange. The basal surfaces of the tendon cells and the terminal ends of the peduncle muscles anchor independently of one another in the collagenous matrix. The peduncle muscles appear to be smooth muscles which contain thin filaments, 5 nm in diameter, and thick filaments, 40-100 nm in diameter, with a faint axial periodicity 12-14 nm. The method of peduncle muscle attachment in the opercular filament is compared with those of other invertebrates.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.     

Morphological analysis of glutaraldehyde-fixed vimentin intermediate filaments and assembly-intermediates by atomic force microscopy

Atomic force microscopy (AFM) was used to study the morphology of vimentin intermediate filaments (IFs) and their assembly intermediates. At each time after initiation of IF assembly in vitro of recombinant mouse vimentin, the sample was fixed with 0.1% glutaraldehyde and then applied to AFM analysis. When mature vimentin IFs were imaged in air on mica, they appeared to have a width of approximately 28 nm, a height of approximately 4 nm and a length of several micrometers. Taking into account the probe tip's distortion effect, the exact width was evaluated to be approximately 25 nm, suggesting that the filaments flatten on the substrate rather than be cylindrical with a diameter of approximately 10 nm. Vimentin IFs in air clearly demonstrated approximately 21-nm repeating patterns along the filament axis. The three-dimensional profiles of vimentin IFs indicated that the characteristic patterns were presented by repeating segments with a convex surface. The repeating patterns close to 21 nm were also observed by AFM analysis in a physiological solution condition, suggesting that the segments along the filaments are an intrinsic substructure of vimentin IFs. In the course of IF assembly, assembly intermediates were analyzed in air. Many short filaments with a full-width and an apparent length of approximately 78 nm (evaluated length approximately 69 nm) were observed immediately after initiation of the assembly reaction. Interestingly, the short full-width filaments appeared to be composed of the four segments. Further incubation enabled the short full-width filaments to anneal longitudinally into longer filaments with a distinct elongation step of approximately 40 nm, which corresponds to the length of the two segments. To explain these observations, we propose a vimentin IF formation model in which vimentin dimers are supercoiling around the filament axis.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.