Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima

An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.     

Ultrastructural study of long spacing collagen fibres and basal lamina in malignant schwannoma

It was found by electron microscopy that extracellular darkly stained materials (DSM) observed abundantly in a case of malignant schwannoma were closely related to both basal lamina and fibrous long spacing collagen (FLS). The FLS were characterized by the cross bands with a 95 nm periodicity, and longitudinally aligned filaments, 9 nm in diameter, while DSM consisted of amorphous material, and 9 nm filaments. The filaments in DSM and FLS were similar in diameter and morphology to reticular fibres in basal laminae. The DSM were continuous with both dark bands of FLS and basal laminae. These results indicate that basal laminae may be the common origin of DSM and FLS. Ultrastructural features of longitudinal, transverse and oblique sections were described.     

Vitrified articular cartilage reveals novel ultra-structural features respecting extracellular matrix architecture

 The quality of cryosections prepared from high pressure frozen bovine articular cartilage has been recently evaluated by systematic electron diffraction analysis, and vitrification found to be zone-dependent. The lower radial layer was optimally frozen throughout the entire section thickness (150 μm), whereas in the upper radial, transitional and superficial layers this was achieved down to a depth of only approximately 5–50 μm. These differences were found to correlate proportionally with proteoglycan concentration and inversely with water content. In the current investigation, extracellular matrix ultrastructure was examined in high pressure frozen material (derived from the lower radial zone of young adult bovine articular cartilage), by both cryoelectron microscopy of cryosections and by conventional transmission electron microscopy of freeze-substituted and embedded samples. Several novel features were revealed, in particular, the existence of a fine filamentous network; this consisted of elements 10–15 nm in diameter and with a regular cross-banded structure similar to that characterising collagen fibrils. These filaments were encountered throughout the entire extracellular space, even within the pericellular region, which is generally believed to be free of filamentous or fibrillar components. The proteoglycan-rich interfibrillar/filamentous space manifested a fine granular appearance, there being no evidence of the reticular network previously seen in suboptimally frozen material. Accepted: 28 June 1996     

The head cartilage of cephalopods. I. Architecture and ultrastructure of the extracellular matrix

The morphology of head cartilage of the cephalopods Sepia officinalis and Octopus vulgaris has been studied on samples fixed and embedded for light- and electron microscopy and on fresh frozen sections viewed by polarizing microscopy. The organization of extracellular matrix (ECM) varies in different regions of the head cartilage. Superficial zones are made up of densely packed collagenous laminae parallel to the cartilage surface, while radially arranged laminae form a deeper zone where territorial and interterritorial areas are present. A compact arrangement of banded collagen fibrils (10-25 nm in diameter) forms the laminae of the superficial zones and of the interterritorial areas; a loose three-dimensional network of fibrils (10-20 nm) with many proteoglycan aggregates forms the territorial areas. A pericellular matrix surrounds the bodies of extremely branched territorial chondrocytes. Peculiar anchoring devices (ADs) are dispersed with variable orientation within the ECM. A perichondrium is present, but often connectival and muscular bundles are fused with the superficial layers of cartilage. Some vessels were also observed within the superficial inner zone and clusters of hemocyanin molecules were demonstrated both in the ECM and in some cells. The cephalopod head cartilage seems to share some morphological characteristics with both hyaline cartilage and bone tissue of vertebrates.     

Three-dimensional organization of the extracellular matrix secreted by cultured rat smooth muscle cells

Summary Specific interactions between cells and the extracellular matrix (ECM) in which they are embedded play a vital role in tissue organization. In recent years, many of the individual components of the extracellular matrix have been isolated and their molecular structures elucidated, but the detailed topography of most extracellular matrices, as they are deposited by cells, is still largely unknown. In this study, the insoluble extracellular matrix produced by cultured rat vascular smooth muscle cells has been characterized morphologically using high-resolution electron microscopy of rotary platinum replicas. These cells grew as flat sheets in culture, secreting their matrix laterally and basally. The matrix was composed of a cross-linked fibrillar meshwork. Some fine fibers (10 to 15 nm in diameter) were naked, but most of the filamentous mesh was covered with coarse granular material. Limited digestion with trypsin or pancreatic elastase removed most of this coating, indicating that the granules were glycoproteins and proteoglycans. Another subset of matrix fibrils (20 to 40 nm in diameter) was identified as type I collagen by direct comparison with purified bovine skin collagen. In addition to exposing the underlying filamentous substructure of the matrix, protease treatment also revealed large, straight fiber bundles and globules of amorphous material suspended in the filamentous web. This novel view of a complex matrix promises to provide spatial information that will be useful in future studies of cell interactions with the ECM. These studies were supported in part by NIH Biomedical Research Support grant S07-RR-05684.     

The ultrastructure of the byssal apparatus of a mussel. V. Localization of collagenic and elastic components in the threads

Ultrastructural and cytochemical studies have been carried out on the proximal part of byssus threads (TPP) in an attempt to localize collagenic and elastic components. The results show that TPP autoclaving followed by hot alkali treatment causes the extraction of about two-thirds of hydroxyproline and the parallel removal of most of the matrix, leaving filaments unaffected. Moreover the results of the staining reactions signaletic for elastic tissues indicate that TPP filaments contain glycoproteins with a reactivity similar to that of many invertebrate elastic tissues. On the basis of these morphological findings, it seems reasonable to suggest that collagen may be located in TPP matrix, while filaments could be responsible for the elastic properties.     

Electron microscope observations on the submicroscopic organization of the retinal rods

The submicroscopic organization of the retinal rods of the rabbit has been studied with high resolution electron microscopy in thin longitudinal and cross-sections. The outer rod segment consists of a stack of flattened sacs or cisternae each of them limited by a thin homogeneous membrane of about 30 A. The membrane of the rod sacs is attached to the surface membrane and is also in continuity with short tubular stalks of about 100 to 150 A which apparently end in relation with the connecting cilium. The bundle of filaments that constitute the connection between the outer and the inner segments is described under the name of connecting cilium. This fibrous component has a structure that is very similar to that of the cilium. It shows 9 pairs of peripheral filaments of about 160 A in diameter, a matrix material, and a surface membrane. Very infrequently two central single filaments are observed. The connecting cilium has a typical basal body in the inner segment; its distal end penetrates the outer segment, where it establishes some structural relation to the rod sacs. The relationships and submicroscopic organization of the connecting cilium were studied in longitudinal and in cross-sections passing at different levels of the rod segments. The inner rod segment shows two distinct regions: a distal and a proximal one. The distal region, corresponding to the ellipsoid of classical histology is mainly composed of longitudinally packed mitochondria. It also contains the basal body of the cilium, vacuoles of the endoplasmic reticulum, dense particles, and intervening matrix with very fine filaments. In the proximal region of the inner segment the mitochondria are lacking and within the matrix it is possible to recognize elements of the Golgi complex, vacuoles of the endoplasmic reticulum, dense particles and numerous neuroprotofibrils of 160 to 200 A in diameter which collect and form a definite bundle at the exit of the rod fiber. The interpretation of the connecting fibers as a portion of a cilium and of the outer segment as a differentiation of the distal part of a primitive cilium are discussed. The importance of the continuity of the surface membranes of the outer segment, connecting cilium, and inner segment is emphasized and its possible physiological role is discussed.     

Immunoelectron microscopy of type X collagen: supramolecular forms within embryonic chick cartilage

To determine the supramolecular forms in which avian type X collagen molecules assemble within the matrix of hypertrophic cartilage, we performed immunoelectron microscopy with colloidal gold-labeled monoclonal antibodies. In addition double-labeled analyses were performed for the molecule and type II collagen, employing two monoclonal antibodies attached to different size gold particles. Both in situ limb cartilages and the extracellular matrix of chondrocyte cultures were examined. We observed in both systems that the type X collagen is present in two forms. One is as fine filaments (less than 5 nm in diameter) within mats which are found predominantly in the pericellular matrix of the hypertrophic chondrocytes. The second form is in association with the fibrils (10-20 nm in diameter) which also react with the antibody for type II collagen. It seems that the filamentous mats represent a form in which the type X collagen is initially secreted from the cell. The type X associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting type II/IX/XI fibrils. The data are consistent with our previously proposed hypothesis that type X collagen is involved in, and perhaps even "targets," certain matrix components for degradation and removal.     

Zebra mussel adhesion: structure of the byssal adhesive apparatus in the freshwater mussel, Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) owes a large part of its success as an invasive species to its ability to attach to a wide variety of substrates. As in marine mussels, this attachment is achieved by a proteinaceous byssus, a series of threads joined at a stem that connect the mussel to adhesive plaques secreted onto the substrate. Although the zebra mussel byssus is superficially similar to marine mussels, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we present an ultrastructural examination of the zebra mussel byssus, with emphasis on interfaces that are critical to its adhesive function. By examining the attached plaques, we show that adhesion is mediated by a uniform electron dense layer on the underside of the plaque. This layer is only 10-20 nm thick and makes direct and continuous contact with the substrate. The plaque itself is fibrous, and curiously can exhibit either a dense or porous morphology. In zebra mussels, a graded interface between the animal and the substrate mussels is achieved by interdigitation of uniform threads with the stem, in contrast to marine mussels, where the threads themselves are non-uniform. Our observations of several novel aspects of zebra mussel byssal ultrastructure may have important implications not only for preventing biofouling by the zebra mussel, but for the development of new bioadhesives as well.     

The ultrastructural organization of the basement membrane of Bowman's capsule in the rat renal corpuscle

Summary The basement membrane of Bowman's capsule (BCBM) of the rat was studied by means of a modified tissue-preservation technique for transmission electron microscopy, which avoids the usual thorough fixation in OsO₄ and applies tannic acid and uranyl acetate for staining (Sakai et al. 1986). At most sites the BCBM is multilayered, consisting of one to seven dense layers separated by electron-lucent layers. The latter, which can be termed laminae rarae, contain fine filaments which connect the dense layers to each other and the innermost dense layer to the basal cell membrane of the parietal epithelium. The laminae densae are basically composed of fine filaments arranged in an anastomosing pattern. Individual filaments ranging from 5 to 15 nm in diameter, combine to form filament bundles up to 100 nm in thickness and 1 to 2 m in length. Within a dense layer, filaments and filamentous bundles are oriented mainly in the same direction. Often the inner dense layers do not form a continuous sheet, and the filamentous bundles are arranged in anastomosing or spiral patterns to form a ribbon-like structure that we call a microligament. These microligaments are often embedded in basal furrows of the parietal epithelium and are best developed around the vascular pole. Intracellular actin bundles of the parietal cells are regularly associated with these extracellular ribbon-like structures of the basement membrane. In conclusion, the BCBM has an unusual structure: the laminae densae are characterized by their filamentous nature and are arranged in different patterns, i.e. as a multilayered mat and as microligaments.Fellow of the Deutscher Akademischer Austauschdienst     

An ultrastructural study of byssus stem formation in Mytilus californianus

In Mytilus californianus, root lamellae of the byssus stem are formed by two morphologically distinct exocrine cell types. Type 1 cells contain large ellipsoid granules which are ultrastructurally identical to those of the collagen gland associated with byssus thread formation: these granules are secreted only at the base of the stem generator. Type 2 cells contain small cylindroid granules which are secreted only from the lateral surfaces of generator septa. The resultant matrix is biphasic because the two secretions are incompletely mixed. Lamellar sheets of matrix are propelled outward by the action of cilia and are molded into a cylinder at the neck region of the stem. However, the stem retains a lamellar pattern. Byssus threads are attached to the stem by flattened rings formed from thread material which is secreted into the cervical crevice surrounding the neck. The microanatomy of the stem forming region is described and a new term, “stem generator,” is proposed for this organ.     

Collagen deposition on a preformed grid

Appearance of collagen fibrils in the cuticle was seen by electron microscopy to be preceded by fonnation of a finely filamentous matrix material. At first, the fine filaments of the matrix are unorganized. However, signs of orthogonal ordering soon appear in the most superficial portion of the cuticle, and subsequently appear more basally and closer to the underlying epidermis. Meanwhile, fibrils of different staining properties and identifiable as collagen begin to be deposited in the superficial portion of the cuticle, the same region which first showed organized fine filaments. Then, like the fine filaments before them, the collagen fibrils polymerize more basally. Collagen appears to polymerize on the preformed skeleton of fine filaments as though the fine filaments caused the collagen to assemble. Neither the polymerization nor ordering of collagen fibrils seems to require direct cellular intervention but occur first in that portion of the cuticle which is furthest away from the underlying epidermis. The fine filaments may be self ordering, extracellular macromolecules which in turn determine the polymerization of collagen fibrils.     

Ultrastructure of the mesoglea of the sea anemone Nematostella vectensis (Edwardsiidae)

Abstract. Cnidarians have extracellular matrix, or mesoglea, situated between an outer epidermis and an inner gastrodermis. In this article, we describe the ultrastructure of the mesoglea of polyps of Nematostella vectensis during development and regeneration. The column wall of recently metamorphosed polyps had basal laminae composed of a meshwork of thin filaments underlying each epithelium and a network of unstriated thick (20–25 nm in diameter) and thin fibrils (～5 nm) decorated with particulate matter. In juvenile polyps with eight tentacles, the system of thick fibrils was concentrated near the gastrodermis. In the column wall and mesenteries of the adult there were bundles of thick fibrils that ran parallel to the myonemes. In regenerating polyps 2 days after transection, the network of thin fibrils and particulate material as well as the basal lamina largely disappeared in the healing part of the oral, but not aboral, half. In the regenerating portion of the aboral half 1 and 2 days after transection, the bundles of thick fibrils were smaller and less organized, and the basal laminae were thicker than in the column wall of untransected polyps. In both regenerating halves, the general organization of the mesoglea of normal polyps was reattained by 5 days after transection. At all stages the mesoglea contained cellular processes that may belong to amebocytes; nucleated amebocytes with a range of shapes were present in the mesoglea of the column wall and mesenteries of adult polyps. Certain features of the mesoglea of members of N. vectensis and Hydra are similar, especially the ultrastructure of the basal laminae, but the fibrillar systems of these two model cnidarians are different. Temporal and spatial differences in the composition of the mesoglea of N. vectensis point to different roles for its components during development and regeneration.     

Composition and ultrastructure of the byssus of Mytilus edulis

Three regions of the byssus of the marine mussel Mytilus edulis L. are distinct in structural organization at the macroscopic and microscopic level and in amino acid composition. The threads that emanate from the stem at the base of the foot are divided into two regions. The proximal, elastic region has a crimped, densely staining cortex enclosing an interior matrix of spiral fibers, and its amino acid composition reflects protein heterogeneity. The more distal, rigid region has a straight, tubular cortex surrounding an inner matrix of linearly arranged bundles of fibrils and has a composition approximating pure collagen. The plaque, or disc-shaped portion, which mediates attachment to various substrates, is distinguished by a surface matrix of collagen-like fibers similar to those of the thread region and anchored on an inner spongy matrix. Compositional evidence exists for a collagenous component, a catechol-rich protein, and at least one other accessory protein in the plaque.     

Byssus thread strength in the mussel, Mytilus edulis

Mytilus edulis attaches to the substratum by means of a proteinaceous byssus complex. This consists of three portions: a root, embedded in the pedal tissues, a stem, continuous with the root but external to the body and a number of byssus threads attached proximally to the stem and distally to the substratum via adhesive discs. Byssus strength varies seasonally on the shore, in response to changes in wave action (Price, in press). As a decline in byssal attachment strength implies a decline in strength of the constituent threads, a study was undertaken to establish the extent to which byssus thread strength is determined by age. The ultimate tensile stress, ultimate tensile strain and Young's Modulus were measured in threads of known age and length and a stepped regression performed on the results. It was found that age and length correlate significantly with tensile stress and Young's Modulus. Length is a less important influence than age on tensile stress but has a greater effect than age on Young's Modulus. Tensile strain is independent of both length and age.     

Electron microscopy of the nucleocapsid from disrupted Moloney murine leukemia virus and of associated type VI collagen-like filaments.

To analyze the constituents of retroviruses, the Moloney murine leukemia virus was disrupted and observed by dark-field electron microscopy. Virus disruption was achieved by several methods: osmotic shock, freezing-thawing cycles, and exposure to urea up to 4 M, to NaCl up to 1 M, and to Triton X-100. Several components associated with broken Moloney murine leukemia virus were repeatedly found in preparations. These components have been described as rings, thick filaments, chain-like filaments, threads covered with proteins, threads with buckles, and naked threads. A quantitative analysis of the occurrence of these components has been carried out. Among them, the thick filaments composed of a compact helical arrangement of small beads 5 nm in diameter were considered to represent the nucleocapsid. The protease-sensitive buckles found on some threads could be a compact form of the viral RNA associated to the nucleocapsid protein NCp10. The RNase-sensitive naked threads are interpreted as the deproteinized viral RNA itself. The ubiquitous chain-like filaments possess a periodic structure identical to that of polymerized type VI collagen. It is proposed that this adhesive protein is associated with the viral envelope taken from the cell membrane during the budding process of retroviruses.     

Sequential assembly of collagen revealed by atomic force microscopy.

Most polymers which comprise biological filaments assemble by two mechanisms: nucleation and elongation or a sequential, stepwise process involving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filament. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correlated with turbidimetric alterations in the assembly mixture. The assembly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of assembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments approximately 2-6 nm in diameter (> 10 microns in length) many with a conspicuous approximately 67-nm axial period were observed. Occasional mature collagen fibrils with a approximately 67-nm axial repeat were found late in the course of assembly. Our results are consistent with initial end-to-end axial association of monomers to form oligomers followed by lateral association into higher-order filaments. On this basis, there appears to be at least two distinctive types of structural interactions (axial and lateral) which are operative at different levels in the assembly hierarchy of collagen.     

Ultrastructure of the flagellar apparatus of Oxyrrhis marina

Summary— Oxyrrhis marina, like all dinoflagellates, possesses one transverse and one longitudinal flagellum, which show structural differences. The transverse flagellum contains a small fibre, 20 nm in diameter, associated with doublet no.7, whereas the longitudinal flagellum is substantially by a large (200–300 nm) hollows structure closely resembling the paraflagellar rod described by several authors in kinetoplastidae and in euglenoids. This structure is made up of a hemicylindrical network of filaments which are often linked on one side to the outer doublet no. 4, and on the other side to a dense plate. Another thinner filamentous network closes this hemicyclinder. In cross-section, the wall of this structure is made up of 8 filaments 2–4 nm in diameter that show a thicker periodic structure. In longitudinal section the same filaments appear arranged in periodic rhombus meshes or a helicoidal pattern, depending on the orientation of the section relative to the axoneme.     

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.     

Ultrastructure of superficial mycosidic integuments of Mycobacterium sp.

Cells from pellicle growth of Mycobacterium sp. NQ are enveloped in a mycoside layer which extends outward as long filaments, 5 nm in diameter. Underneath this outer mycosidic casement, ramified ropelike structure, embedded in a dense matrix, overlay the rigid peptidoglycan of the cell wall.