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.     

Connective tissue of rat lung. II: Ultrastructural localization of collagen types III, IV, and VI

We localized collagen types III, IV, and VI in normal rat lung by light and electron immunohistochemistry. Type IV collagen was present in every basement membrane examined and was absent from all other structures. Although types III and VI had a similar distribution, being present in the interstitium of major airways, blood vessels, and alveolar septa, as in other organs, they had different morphologies. Type III collagen formed beaded fibers, 15-20 nm in diameter, whereas type VI collagen formed fine filaments, 5-10 nm in diameter. Both collagen types were found exclusively in the interstitium, often associated with thick (30-35 nm) cross-banded type I collagen fibers. Occasionally, type III fibers and type VI filaments could be found bridging from the interstitium to the adventitial aspect of some basement membranes. Furthermore, the association of collagen type VI with types I and III and basement membranes suggests that type VI may contribute to integration of the various components of the pulmonary extracellular matrix into a functional unit.     

Reprint of "Structural correlation between collagen VI microfibrils and collagen VI banded aggregates" [J. Struct. Biol. 154 (2006) 312-326

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.     

Structural correlation between collagen VI microfibrils and collagen VI banded aggregates

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.     

Type VI collagen: immunohistochemical identification as a filamentous component of the extracellular matrix of the developing avian corneal stroma

Selected stages of the developing chicken cornea have been examined for type VI collagen, employing monoclonal antibodies specific for this molecule. By immunofluorescence, the molecule is not detectable in 5 1/2 day corneas, a time at which the epithelial-derived, acellular primary stroma is the only corneal matrix present. One day later, the presumptive stromal fibroblasts have invaded this stroma and have initiated synthesis of the secondary (mature) stroma. By that time, a strong fluorescent signal for the type VI collagen molecule is detectable throughout the stroma. It is present in all subsequent ages examined. The molecule is not restricted to the cornea, and is present in most stromal matrices examined, including those of the sclera, eyelid, and nictitating membrane. Immunoelectron microscopy was also performed, utilizing a colloidal gold-labeled secondary antibody. These data show that the type VI collagen is not a component of the striated collagen fibrils, but instead is assembled in the form of thin filaments. The monoclonal antibody bound to the filaments at periodic intervals of about 100 nm.     

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.     

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.     

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.     

Distribution of type III collagen in the pulp parenchyma of the human developing tooth

Summary The distribution of collagen type III throughout the pulp tissue from human developing tooth was studied using specific antibodies, immunofluorescence as well as immuno-peroxidase labelling for electron microscopy.Our results indicate that type III and type I collagen are present in the pulp. The staining intensity seems to correlate with the relatively high proportions of type III collagen biochemically found in pulp. In addition, type III collagen and reticulin fibres are similarly distributed, except that the Von Korff fibres were never detected with anti-type III collagen antibodies. Correspondingly, at the ultrastructural level, type III collagen appears as fine, branched filaments or electron dense material distributed throughout the tissue and particularly in close association with the plasma membrane of pulp fibroblasts. In contrast, type I collagen appears as typical coarse cross banded fibres.     

Ultrastructural Basis of the Tension Increase in Sea-Urchin Eggs Prior to Cytokinesis

Cortices of sea-urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70–90 Å in diameter appeared in the cell cortex forming a thin mesh-work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork-filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

Myosin VI stabilizes an actin network during Drosophila spermatid individualization

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.     

Extracellular and intracellular degradation of collagen by trophoblast giant cells in acute fasted mice examined by electron microscopy

The fine structure of trophoblast giant cells and their interaction with collagen at the antimesometrial region on the 9th day of pregnancy was examined in fed and acute fasted mice. Collagen fibrils and filamentous aggregates (disintegrating collagen fibrils) were observed in the extracellular space. Three types of intracellular vacuoles containing collagen fibrils were present: vacuole type A exhibited typical cross-banded collagen immersed in finely granular electron-translucent material; and vacuoles type B and C showed electron-opaque granular material containing, respectively, faint cross-banded collagen and narrow clear stripes often with faint periodicity. In fed animals vacuoles type B were absent and the others were less evident.Only fasted animals showed extracellular acid phosphatase activity on collagen fibrils, filamentous aggregates and confined regions of the extracellular space. Intracellular acid phosphatase activity was observed in vacuoles type B and in lysosomes.The results indicate that trophoblast giant cells are capable of breaking down extracellular collagen and also of internalizing collagen for intracellular degradation. It is likely that these events are part of the process of invasion of the uterine wall. However, in fasted mice, collagen breakdown is more pronounced, and it may therefore contribute to the provision of amino acids and other nutrients for the undernourished fetus.     

Two type XII-like collagens localize to the surface of banded collagen fibrils

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.     

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.     

Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N- and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.     

Type VI collagen is a major component of the human cornea

Collagen type VI is shown to be present in the human cornea. This finding is based on comparative peptide mapping relative to type VI collagen isolated from placenta and on immunoblotting using antibodies specific for human type VI collagen. Scanning of polyacrylamide gels indicates that type VI collagen comprises as much as one quarter of the dry weight of the cornea. Indirect immunofluorescence shows this collagen to be distributed throughout the corneal stroma. Thus, type VI collagen must be considered a major component of the extracellular matrix of the human cornea.     

Über bandartige Strukturen in den Chromozentren vonLupinus polyphyllus

In the nuclei ofLupinus polyphyllus strongly marked chromocentres occur. In electron micrographs of anther cell nuclei these chromocentres appear either as a homogeneous network or subdivided into two distinct regions, a network-like (NR) and a banded one (GR). In both a 100 Å fiber is the basic unit. The GR is composed of 4 to 6 parallely arranged electron dense bands (240–280 Å wide) and interbands (260–300 Å wide) of low electron density. They appear to correspond to a cylindrical structure with disc-like components and connective sections. These observations are discussed in relation to chromosome structure during interphase and mitosis.

Herrn Prof. Dr. L.Geitler zur Vollendung seines 80. Lebensjahres gewidmet.     

Corneal cell-matrix interactions: type VI collagen promotes adhesion and spreading of corneal fibroblasts.

Type VI collagen is a nonfibrillar collagen present as a network throughout the chick secondary stroma. Immunolocalization of type VI collagen both in the chick corneal stroma and in other systems demonstrates that type VI collagen is present associated with cells and between striated fibrils. We hypothesize that type VI collagen may function in cell-matrix interactions important in corneal development. To examine this possibility, we have isolated and characterized bovine corneal type VI collagen and determined that the chain composition and morphology of type VI collagen isolated from cornea is similar to that isolated from other sources. The tissue form of type VI collagen was localized to filaments forming a network around fibrils and close to corneal fibroblasts. We then analyzed relative attachment and spreading on type VI collagen as compared to the other collagens present in the secondary stroma, and found that although corneal fibroblasts attach equally well to type VI and type I collagen, cells spread to a much greater extent on type VI collagen. Although corneal fibroblasts do have an RGD-dependent receptor which functions during adhesion to fibronectin, attachment to type VI collagen is RGD-independent unless the molecule is denatured. Blocking of the RGD-dependent receptor with soluble RGD peptides results in no change in attachment or spreading. These data imply a role for type VI collagen in cell-matrix interactions during corneal stroma development.     

Distribution of type III collagen in the pulp parenchyma of the human developing tooth. Light and electron microscope immunotyping

The distribution of collagen type III throughout the pulp tissue from human developing tooth was studied using specific antibodies, immuno-fluorescence as well as immuno-peroxidase labelling for electron microscopy. Our results indicate that type III and type I collagen are present in the pulp. The staining intensity seems to correlate with the relatively high proportions of type III collagen biochemically found in pulp. In addition, type III collagen and reticulin fibres are similarly distributed, except that the Von Korff fibres were never detected with anti-type III collagen antibodies. Correspondingly, at the ultrastructural level, type III collagen appears as fine, branched filaments or electron dense material distributed throughout the tissue and particularly in close association with the plasma membrane of pulp fibroblasts. In contrast, type I collagen appears as typical coarse cross banded fibres.