Fibrillar Array in the Cell Wall of a Gliding Filamentous Cyanobacterium

The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.     

Ultrastructure of the vitreous body of the rabbit

Examination in the scanning and the transmission electron microscope showed three morphologically and structurally different types of cells in the vitreous body of the healthy rabbit eye: 1. cells with numerous cytoplasm processes, whose high metabolic activity is represented by the presence of a large number of organelles and which are capable of synthesizing fibrillar material; 2. elongate cells with a flattened nucleus, with long, narrow cytoplasm processes arising from both their poles and with only a few organelles in their cytoplasm; 3. large spherical cells with structureless contents, whose nucleus and few organelles are situated below the cell membrane. The organized component of the intercellular matter of the rabbit vitreous body is composed of collagen fibrils with a very variable diameter (24-180 nm), The collagen fibrils form the basis of the three-dimensional skeleton of the intercellular matter of the vitreous body.     

ELECTRON-OPAQUE FIBRILS AND GRANULES IN AND BETWEEN THE CELL WALLS OF HIGHER PLANTS

The components of higher-plant cell walls which become electron-opaque after staining with ruthenium-osmium were studied by electron microscopy. A fibrillar material which absorbs this stain is a major wall constituent in the root epidermal cells of carrot and morning glory. In both form and size, these fibrils resemble those found on the surface of suspension-cultured cells of the same species Some cells of woody species show an irregular distribution of electron-opaque material in the cell wall matrix and middle lamella. This material, which has an amorphous appearance with many electron stains, is shown by ruthenium-osmium staining to be an aggregate of discrete granules, 150–220 A in diameter. These observations are not consistent with the concept of the cell wall matrix and middle lamella as an amorphous, uniform gel     

FURTHER CYTOLOGICAL STUDIES OF A PERIODIC ACID-SCHIFF'S SUBSTANCE IN THE OVULES OF PASPALUM ORBICULARE AND P. LONGIFOLIUM

A periodic acid-Schiff's substance present in the micropylar end of the ovules of Paspalum orbiculare and P. longifolium was further studied by light and electron microscopy of glutaraldehyde-osmium-fixed and freeze-substituted, osmium-fixed tissues. The PAS substance is water soluble and is found in intercellular spaces between the nucellus and inner integument, the inner and outer integuments, the outer integument and ovary wall, and in the micropyle. Structurally the substance consists of fibrils embedded in a dense, amorphous matrix and may be associated with membranous structures in special layers between the plasmalemma and the cell wall in nucellar and integumentary cells. Part of the water soluble substance is believed to be secreted from the nucellar and integumentary cells. A large amount of this substance may be formed as a result of the dissolution of about one third of the distal micopylar portion of the outer integument prior to anthesis. Many of the electron-dense fibrils seem to be fibrillar intercellular substances and others appear to originate from the cell walls, including the cuticle. Both the matrix and the fibrils may be chemically heterogeneous and together form a mucilagenous substance which may facilitate the final growth of pollen tubes in these two species.     

Factor VIII-related antigen : A pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of endothelial cells as well.In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with M_r 220–240; 180; 160; 80 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.     

Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus.

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.     

An electron microscopic study of non-fixed and non-dehydrated normal human stratum corneum

Summary This is an electron microscopic study of non-fixed and non-dehydrated normal human stratum corneum from the lumbar region.Non-stained sections have a low contrast. In sections examined 3 days after skin biopsy the cytoplasm of the cells shows a uniform contrast or exhibits dark and light areas. A single layer delimits the cytoplasm from the intercellular space. The latter is partly filled out with substance.In sections stained 2 to 4 days after skin biopsy the fibrils are distinct. On the basis of the variations in their opacity and ultrastructure three types of horny cells are clearly distinguishable. In cells of type 1 intensely stained keratohyalin and less opaque fibrillar substance occur. A distinct keratin pattern is not found. In cells of type 2 the fibrils show areas with distinct kerytohyalin and keratin pattern and transitional phases between these two stages of fibrillar differentiation. The keratin pattern representing the final stage of the fibrillar differentiation process is visualized through a successive discoloration of the filaments, whereas the interfilamentous substance retains the opacity of the keratohyalin. In cells of type 3 the entire fibrillar substance exhibits a keratin pattern. This consists of less opaque filaments with a diameter of 74 Å. The septa representing the interfilamentous substance are estimated as 30 Å at their thinnest points. These observations of the fibrils are completely comparable to the findings in fixed and dehydrated normal human stratum corneum.In sections stained particularly more than 18 days after skin biopsy the fibrils exhibit pronounced changes in their staining properties with concomitant decrease in distinctness or a complete extinction of the keratin pattern.The observations of the modified plasma membrane and the intercellular space in stained sections correspond to the findings in fixed and dehydrated normal human stratum corneum. The modified plasma membrane and the structures in the intercellular space appear with equal distinctness, whether the sections are stained 2 to 4, 6 to 12 or 14 to 21 days after skin biopsy.This investigation was supported by grants from the Edvard Welander Foundation and from the Swedish Medical Research Council (B71-12X-2708-03).     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : III. The Surface Layers of Chondrococcus columnaris

An electron microscope study of the myxobacterium Chondrococcus columnaris has revealed the following structures in the peripheral layers of the cells: (1) a plasma membrane, (2) a single dense layer (probably the mucopeptide component of the cell wall), (3) peripheral fibrils, (4) an outer membrane, and (5) a material coating the surfaces of the cells which could be stained with the dye ruthenium red.The ruthenium red-positive material is probably an acid mucopolysaccharide and may be involved in the adhesive properties of the cells. The outer membrane and plasma membrane both have the appearance of unit membranes: an electron-translucent layer sandwiched between two electron-opaque layers. The peripheral fibrils span the gap between the outer membrane and the mucopeptide layer, a distance of about 100 A, and run parallel to each other along the length of the cell. The fibrils appear to be continuous across the ends of the cells. The location of these fibrillar structures suggests that they may play a role in the gliding motility of these bacteria.     

FACIT collagens: diverse molecular bridges in extracellular matrices

The collagens form a large family of proteins. Collagen fibrils, composed of staggered arrays of fibrillar collagen molecules (types I, II, III, V and XI), provide a supporting scaffold for extracellular matrices of connective tissues. The non-fibrillar collagens are less abundant than the fibrillar collagens, but it is becoming clear that they have important functions in the matrix. Recently, a group with unique structural characteristics has been defined and named the FACIT (Fibril-Associated Collagens with Interrupted Triple-helices) group. There is evidence that these collagens may serve as molecular bridges that are important for the organization and stability of extracellular matrices.     

Intercellular matrix in colonies of Candida.

The histochemistry and fine structure of typical colonies of six species of Candida were studied, using a total of 31 clinical isolates. The colonies consisted of viable and degenerate cells which lay in an intercellular matrix. This matrix was made up of amorphous, granular, and fibrillar components, the relative proportions and total amount of which varied from species to species. The cells of all species were surrounded by a zone of homogeneous amorphus material, which may be a highly cross-linked carbohydrate. This separated intact cells from irregularly distributed granular debris derived from the cytoplasm of degenerate cells. Focal cellular degeneration and associated granular debris were present within the colonies of all species and were most common in the surface layers of cells of colonies of C. albicans and C. tropicalis. The large amounts of intercellular matrix in this region formed a surface coat on colonies of these two species. Intercellular strands of cell wall material, and to a lesser extent other membranous elements from degenerate cells, formed a prominent fibrillar meshwork in the colonies of C. albicans and C. tropicalis, but were less common in those of C. pseudotropicalis and C. guilliermondii and seldom seen in those of C. parapsilosis and C. krusei.     

The relationships between ribosomal genes and fibrillar centers in thyroid cells cultivated in vitro

Despite the fact that the fibrillar centers of the nucleolus and the chromosomal nucleolar organisers (NORs) are similarly stained with the NOR-silver technique, there remain some questions about the identification of fibrillar centers as NORs. The distinct delineation of the fibrillar centers in porcine thyroid cells allowed us to determine whether there was a numerical equivalence or correlation between fibrillar centers and NORs. Hybridization in situ and silver staining performed on pig chromosomes showed that pairs 8 and 10 contained rDNA sites. Silver staining of thyroid cells in electron microscopy showed that the fibrillar centers and their surrounding layer of dense fibrils were the sites of silver deposit. Chromatin fibers were demonstrated within the fibrillar centers through the aid of the osmiumammine reaction and with the oxidized diaminobenzidine technique. It was observed that in cultured thyroid cells the fibrillar centers could be identified in the light microscope as argyrophilic spherules, and easily counted. The number of fibrillar centers was variable according to culture conditions. In cells cultured for 5 hr, the mean number of fibrillar centers was 1.7. After 5 days of culture, the number of fibrillar centers increased, reaching a mean value of 5.93. When thyroid cells were stimulated with thyrotropin, the number of fibrillar centers again increased to a mean value of 7.54. These results demonstrate that the relationship between fibrillar centers and NORs is not a simple proportionality: the number of fibrillar centers increases with increased cellular activity. These data imply that in active cells each NOR may pass through several fibrillar centers.     

Scale formation in chrysophycean algae

Summary The two types of scales of the coccolithophorid Hymenomonas (= Cricosphaera) carterae have been studied with the negative staining technique. Both types of scales, including the non-mineralized circular ones and the larger, mineralized elliptical ones are made up of a highly ordered network of radial and concentric fibrils. Both fibrils are ribbon shaped and have dimensions of 10–22×30–75 Å in cross-sections. The margin of the scales is composed of two closely associated fibrillar ribbons which are observed occasionally in edge view. In the elliptical scales, the periphery of the fibrillar network often is obscured by a deposition of the coccolith calcium carbonate. The substructure of the fibrils, the occurrence of kinking sites, and the fibrillar resistance to alkaline and weak acid treatments strongly resemble the scales of Pleurochrysis scherffelii. In relation to the recent findings of Brown and co-workers (Brown et al., 1969, 1970) on scale formation processes, it is hypothesized that the fibrillar network of the scales of both species is structurally and chemically identical, and that the fibrillar material consists of a cellulose-like polysaccharide.Supported by the Deutsche Forschungsgemeinschaft (to W. W. F.) and NSF Grant # GB 23047 (to R. M. B.).     

The fibrous system of the extracellular matrix of Podocoryne carnea and its degradation by the subumbrellar plate endoderm demonstrated by a monoclonal antibody

Homogenized fragments of crude umbrellar material of the hydromedusa Podocoryne carnea was injected into BALB/c mice. The immunization resulted in the isolation of a monoclonal antibody designated 3A1 which specifically binds to fibrils in the mesogloeal extracellular matrix (ECM) of hydromedusae. In vivo, the architecture and the ultrastructure of the fibrous system in the outer mesogloea (outer ECM) of Podocoryne carnea, and its degradation under in vitro conditions have been described by morphological and immunological criteria. In vivo, 120-150 A thick, striated fibrils (with periodicities up to 50 nm) form a threedimensional network which fills in the entire outer ECM. Vertical fibers (up to 150 nm in diameter) penetrate the three-dimensional network and branch at the subumbrellar and the exumbrellar side. The vertical fibers show uniform distribution over the entire outer ECM. The branches impinge on a dense matrix (about 30 nm in thickness) covering the exumbrellar and subumbrellar surface. In vitro, the fibrillar system does not alter in its basic pattern, neither in isolated outer ECM, nor in portions of outer ECM which is either covered by the exumbrella, or which is attached to both: the exumbrella at the outside, and the subumbrellar plate endoderm at the inner side. After removal of the exumbrellar cells in the latter portions, a characteristic pattern of selective degradation of the outer ECM by the endodermal cells is observed. This process involves three distinct steps: an initial extracellular condensation within the ECM fibrillar network, followed by intercellular internalization of the fibrillar elements and subsequent endocytosis of ECM material. The first step immediately follows the removeal procedure of the exumbrellar cells and is completed within minutes. This process cannot be interrupted by dihydrocytochalasin B (H(2)CB). The second step lasts 24-48 hr, is mediated by cell mechanisms, and can be stopped by H(2)CB. The third step is a slow process (of up to 14 days). It involves intercellular degradation of fibrillar material, endocytosis, and completion of digestion within lysosomes.     

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.     

Periodic fibrillar material in intracellular vesicles and in electron-dense bodies in chondrocytes of rat costal and tracheal cartilage at various ages.

The occurrence of intracellular fibrillar material (frequently banded) has been studied in normal costal and tracheal chondrocytes of rats at various ages ranging from 1 to 90 days. The study methods have included digestion with collagenase, electron histochemical techniques and routine electron microscopy. Banded fibrillar material has been observed intracellularly in vesicles or in electron-dense bodies in perichondrial and subperichondrial chondrocytes from rats of all ages. These fibrils and extracellular collagen fibrils are partially and equally degradable by collagenase, they are positive after staining with phosphotungstic acid or with silver nitrate methenamine, and their lucency corresponds with that of collagen when they are stained only with lead citrate. They have not been observed in intracellular clefts. They, therefore, seem to be formed intracellularly and to be exocytosed subsequently. Large vesicles and electron-dense bodies seem to be derived from Golgi saccules. A mechanism whereby banded intracellular fibrils could be formed from tropocollagen molecules is postulated. The frequency of occurrence and the diameter of intracellular fibrils seems to increase with increasing age.     

Fibrillar structure and charge determine the interaction of polyglutamine protein aggregates with the cell surface

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces.     

A CYTOCHEMICAL AND RADIOAUTOGRAPHIC STUDY OF HUMAN TISSUE CULTURE CELL NUCLEOLI

Fine structural aspects of human tissue culture cell nucleoli were studied by cytochemical and radioautographic methods. Ribonuclease and pepsin digestions were carried out on glutaraldehyde-fixed cells that, in some instances, were labeled with thymidine-³H prior to digestion. Double digestion by ribonuclease and pepsin revealed a fine fibrillar reticulum that appears to be the supportive structure of nucleolonemal threads. The nature of the reticulum remains to be determined. The question of whether it may represent a dispersed form of chromatin was raised. Structural findings suggested such an hypothesis but the results of radioautographic studies do not support it. The reticulum showed a striking absence of radioactive labeling following a 3 hr incorporation of thymidine-³H. Only few silver grains were observed occasionally in the fibrillar nucleolonema that may or may not be significant. The radioautographic results are believed to be inconclusive for the various reasons discussed. The possibility that the reticulum is composed of proteins has to be considered. It appears that basic proteins can resist pepsin digestion in aldehyde-fixed cells. Individual chromatin fibrils were found to be associated with the nucleolar reticulum. It is possible that these alone represent the dispersed genetically active chromatin of nucleoli.     

Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.     

Intercellular accumulation of type V collagen fibrils in accordance with cell aggregation

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.